First Reported Case of Spontaneous Hermaphrodism in Female Date Palm (Phoenix Dactylifera L.), Cv ‘Alligue’

The Journal of Horticultural Science and Biotechnology

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First reported case of spontaneous hermaphrodism in female date palm (Phoenix dactylifera L.), cv ‘Alligue’

Ahmed Othmani, Myriam Collin, Amel Sellemi, Shri Mohan Jain, Noureddine Drira & Frédérique Aberlenc

To cite this article: Ahmed Othmani, Myriam Collin, Amel Sellemi, Shri Mohan Jain, Noureddine Drira & Frédérique Aberlenc (2017): First reported case of spontaneous hermaphrodism in female date palm (Phoenix dactylifera L.), cv ‘Alligue’, The Journal of Horticultural Science and Biotechnology To link to this article: http://dx.doi.org/10.1080/14620316.2017.1280379

Published online: 28 Feb 2017.

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Download by: [othmani ahmed] Date: 10 March 2017, At: 01:59 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY, 2017 http://dx.doi.org/10.1080/14620316.2017.1280379

First reported case of spontaneous hermaphrodism in female date palm (Phoenix dactylifera L.), cv ‘Alligue’ Ahmed Othmania, Myriam Collinb, Amel Sellemia, Shri Mohan Jainc, Noureddine Drirad and Frédérique Aberlencb aLaboratoire de culture in vitro, Centre Régional de Recherches en Agriculture Oasiennes, Degache, Tunisia; bUnité Mixte de Recherche DIADE (diversité, adaptation, développement des plantes), IRD Montpellier, France; cDepartment of Agricultural Sciences, University of Helsinki, Helsinki, Finland; dLaboratoire des Biotechnologies Végétales Appliquées à l’Amélioration des Cultures, Faculté des Sciences de Sfax, Sfax, Tunisia

ABSTRACT ARTICLE HISTORY The date palm (Phoenix dactylifera L.) is dioecious with male flowers deficient in functional Accepted 4 January 2017 gynoecium and female flowers deficient in functional androecium borne on separate palms. KEYWORDS The presence of male and female flowers on the same plant, a phenomenon known as Date palm; cv Alligue; monoecy, is unusual in male date palms. flowers; hermaphroditism; This study reports for the first time on hermaphrodite (bisexual) flowers borne by two female inflorescence; date palms, ‘Alligue’, that were found growing in an open field in Degache, southern Tunisia. The parthenocarpy; rachilla observations on these two female palms were compared with hermaphrodite male date palms growing in the same location. Hermaphrodite female date palm inflorescence branches bear female flowers predominantly near their base, in contrast to the hermaphrodite flowers that are found primarily toward their upper part. The position of the hermaphrodite flowers in hermaphrodite male date palms is reversed: inflorescence branches bear male flowers toward the upper part while the hermaphrodite flowers are found at the base. Histological examination of female hermaphrodite flowers revealed that they had three carpels and 1–6 stamens. Hermaphrodite flowers on male plants were usually also composed of three carpels of variable size, and six stamens. Hermaphrodite flowers on both female and male palm trees turn generally into parthenocarpic fruits. The present data support the theory that dioecious plants are derived from a common hermaphrodite ancestor. Floral hermaphroditism in date palm should be investigated in relation to the in planta self- fertilization process to identify sex markers and genes that control sex organ development.

Introduction A typical date palm inflorescence consists of numer- ous unbranched rachillae spirally arranged on a single The genus Phoenix or pinnate-leaved palms is comprised flattened rachis. Both male and female flowers are ses- of 17–19 dioecious species (Carpenter & Ream, 1976), sile on the rachilla and enclosed in its early stages in a native to the tropical or subtropical regions of Africa or hard, fibrous envelope known as a spathe that protects southern Asia (Old World), including Phoenix dactyli- the delicate flowers from the intense heat and sunlight fera L. (Munier, 1973).Thelatteristhemosteconomic- during the early stage of flower development ally important palm next to the coconut palm and is the (ChihCheng & Robert, 2007). The spathe splits open mainstay of agriculture in oasis zones where many other as the flowers mature, exposing the entire inflorescence food crops can be grown in combination. Recently, ser- for pollination (Zaid & De Wet, 2002). The spathes are ious efforts have been geared toward understanding of shorter and wider in male compared to female trees. the genetic basis and the sex determination mechanism Generally, the female inflorescence has fewer florets in date palm, with the aim of identifying sex before per rachilla compared to that of the male inflorescence. reproductive age through the employment of molecular Each spikelet carries a large number of tiny flowers, as marker tools (Al-Dous et al., 2011;Cherifetal.,2013; many as 8000–10,000 in female and even more in male Siljak-Yakovlev et al., 1996). These studies indicate that inflorescence (Chandler, 1958). Zaid and De Wet (2002) date palm is one of the rare monocotyledonous dioecious reported that the female flower has a diameter of about species that employs heteromorphic XY sex chromo- 3–4 mm and has rudimentary stamens and three carpels somes of gender inheritance. DeMason and Tisserat closely pressed together, and the ovary is superior (hypo- (1980) were the first to report that, in certain cases, gynous). The three sepals and three petals are united some male date palm trees give rise to apparent bisexual together so that only tips diverge. On opening, the female flowers. flowers show more yellow color while the male ones

CONTACT Ahmed Othmani [email protected] Laboratoire de culture in vitro, Centre Régional de Recherches en Agriculture Oasiennes, P.O. Box 2260, Degache, Tunisia © 2017 The Journal of Horticultural Science & Biotechnology Trust 2 A. OTHMANI ET AL. show white-colored dust, produced on shaking. The spathe anthesis and followed in the course of all their pollen sacs usually open within an hour or two after the phenological stages. This study was repeated 3 times bursting of the spathe. Only one ovule per flower is during 3 successive years (2012, 2013, and 2014). fertilized, leading to the development of one carpel which in turn gives a fruit called a date. The other ovules Histological study abort and persist as two brown spots in the calyx of ripe fruits. The male inflorescences are densely packed with For histological analysis, 20 bisexual female flowers were flowers and the rachillae nod at the time of anthesis fixed in fixation buffer (10% paraformaldehyde, 25% −1 (Munier, 1973). glutaraldehyde, 1% caffeine, and 0.1 mol L 21 phos- We are reporting for first time two cases of a phate buffer, and pH 7). Flowers were then dehydrated spontaneous bisexuality in female date palm, cv through a graded ethanol series (30, 50, 70, 80, 90, 95, and Alligue, in Tunisia with the aim to add further infor- 100%) and stored at 4°C. The dehydrated inflorescences mation on this important phenomenon. were transferred to a 50% ethanol:50% butanol solution for 1 h, then to 100% butanol for 2 h, followed by a transfer for 2 days to several months to a 100% butanol Materials and methods solution. For their inclusion, inflorescences were Materials embedded in the resin, Technovit 7100 (Heraeus Kulzer GmbH, Germany) and impregnated with a solution of The vegetal material used in this study consisted of resin and butanol (50%:50%). Then, Hardener II of flowers and fruits at different phenological stages col- Technovit 7100 kit was added to the resin. The blocks ≈ lected from a clone of a ( 45-year-old) female date obtained were stacked with cold-curing resin of ‘ ’ ≈ palm Alligue ,havingthree 30-year-old offshoots Technovit 3040 kit. The resin was allowed to polymerize ‘ ’ (Figure 1); four 25-year-old male date palms ( P7 , for a few hours at room temperature. ‘ ’ ‘ ’ ‘ ’ Abd1 , Abd2 ,and Abd3 );anda27-year-oldbisexual Sections (3.5–4.0 mm thick) were obtained using a ‘ ’ male date palm ( P8 ). The sampled source plants grow in microtome (Microm). Finally, the sections were double- the oasis field of Degache, a region in the southwest of stained with periodic acid (Sigma P-7875) and protein- Tunisia. The bisexual flowers collected from the studied specific Naphthol Blue Black (Sigma N-3005) and then plants were counted in every inflorescence just after observed under a light microscope.

Pollen fertility M Two methods were used. First, inflorescences were covered with craft paper before anthesis to avoid cross- pollination. Secondly, stamens of bisexual flowers were transferred to the flowers of another female date palm variety, ‘Deglet nour’, before anthesis. After this transfer, the latter inflorescence was also covered with craft paper.

Seed germination Germination of date palm seeds requires soaking them in a solution of sodium hypochlorite (20%) for 24 h followed by transfer to an empty Petri dish containing three sterile filter paper disks imbibed by water and placed in air conditioned culture room at 2 1 28 ± 2°C with 16/8 h photoperiod providing 80 μmol − − m 2 s 1 fluorescent light for 3 weeks.

3 Results Structure of inflorescences produced by the bisexual female tree The inflorescences of offshoots 2 and 3 appeared Figure 1. Photo of a clone of date palm variety ‘Alligue’ similar to those produced by ordinary female trees. composed of a mother plant (M) and offshoot 1 producing The rachillae of these inflorescences are divided into only female flowers, and offshoots 2 and 3 producing both two groups: the first one is dominant and includes female and bisexual flowers. Scale bar 20 cm. completely typical female flowers, and the second one THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 3 is composed of both typical female flowers and apparent bisexual flowers (Figure 2). b f Structure of flowers produced by the two bisexual female date palms m Macroscopic scale As described, date palm has dimorphic flowers produced by separate male or female palms. Both female Figure 3. Photo of enlarged typical male flowers (m) pro- and male flowers usually have three sepals in a cupule duced by the male ‘P7’ and bisexual flower (b) and typical at their bases, and three petals. Petals of female (pis- female flowers (f) produced by bisexual female tree (offshoot tillate) flowers are short, thick, imbricate, and welded 3): m: typical male flower, b: bisexual flower and f: typical but those of male (staminate) flowers are long, sepa- female flower. Scale bar 1 cm. rated, sharp, and only slightly imbricate. The female flowers have three separate and erect carpels and six rudimentary stamens (staminodes) that constitute small projections on the floral receptacle between the carpels and petals. However, the male flowers have three small separate carpels (pistillodes) located in the center and surrounded by six stamens which produce abundant pollen and are densely distributed on the rachillae. In the case of offshoots 2 and 3, we detected typical female flowers, and bisexual flowers having petals that resemble in shape and arrangement those of the male flowers (Figures 2 and 3), but intermedi- ate in size between female and male flowers (Figure 4 and Table 1) which is positively correlated to the length of stamens (Figure 5). These flowers can Figure 4. Sepals (s) and petals (p) of female, male and have from one to six stamens but usually three iden- bisexual flowers produced by bisexual female date palm. tical carpels with size inversely proportional to the Scale bar 0.5 cm. number of stamens (Figure 6). The distribution of such flowers is not arbitrary on the rachillae (spike- Table 1. Petals size of male, female, and bisexual flowers lets) but they occupied preferentially its upper part produced by bisexual female date palm. (Figure 7 and Table 2). Male flowers Bisexual flowers Female flowers Sepal length 0.65 ± 0.05 0.40 ± 0.05 0.28 ± 0.05 Microscopic scale Sepal width 0.46 ± 0.05 0.65 ± 0.05 0.85 ± 0.05 Longitudinal sections were carried out on apparent bisexual flowers. Examination of these sections under the light microscope showed morphologically normal male and female organs (Figure 8). Indeed, the stamens are surmounted by two pollen sacs filled with spherical pollen grains (Figure 9(a)) each with generative nucleus and pollen tube nucleus (Figure 9(b)–(e)). 1 As for the gynoecium, it is composed of three carpels b 2 with one ovule each (Figure 10). Every ovule possesses an embryo sac (megaspore) within which the embryo f develops.

3 Structure of inflorescences produced by a bisexual male date palm Figure 2. Photos of a rachilla produced by the male ‘P7’ (1) and Bisexual flowers were also observed on some males rachillae produced by bisexual female tree (offshoot 3): (2) rachilla such as ‘P8’ that flowers twice a year, and more with bisexual (b) and typical female flowers (f), and (3) rachilla precisely at the second flowering that, in some with only typical female flowers. Scale bar 1 cm. years, occurs in the month of June (Table 3), 4 A. OTHMANI ET AL.

a b

cc’

dd e’

Figure 5. Photos demonstrating the positive correlation between length of stamens and that of petals taken from bisexual flowers of female bisexual date palm. (a) Structure of stamen (length: 3 mm). (b) Frontal cutting showing the arrangement of stamens behind petals. (c and c') Bisexual flower with short petals before and after removal of sepals and petals, respectively. (d and d') Bisexual flower with medium-sized petals before and after removal of sepals and petals, respectively. (e and e') Bisexual flower with long petals before and after removal of sepals and petals, respectively. Scale bar 0.5 mm

stamen

carpel

a bc

d e f

Figure 6. Different types of bisexual flowers with three carpels that differ by the number of stamens. a, b, c, d, e, and f with 1, 2, 3, 4, 5, and 6 stamens, respectively. Scale bar 1 mm. necessarily governed by the climate. In some males, Inflorescences of a bisexual male tree such as ‘P8’ all flowers produced whether at first flowering or at are composed of two types, namely typical male second are typically male, for example the males Abd flowers that occur generally at the upper part of the 1, 2, and 3. rachillae and bisexual flowers occurring mostly at THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 5

Figure 7. Rachillae of a bisexual inflorescence of offshoot 2. (a) the upper part, and (b) the lower part. Scale bar 1 cm

Table 2. The percentage and the position of bisexual flowers at inflorescences and rachillae of the bisexual female date palm. Inflorescences with bisexual Bisexual Bisexual rachillae with bisexual flowers at Bisexual rachillae with bisexual flowers at Year flowers (%) flowers (%) their top part (%) their basal part (%) Offshoot 2012 60 33.4 87.4 0 2 2013 50 21.2 83.2 0 2014 33.33 5.3 71.3 0 Offshoot 2012 66.6 30.2 79.3 0 3 2013 50 19.3 67.4 0 2014 20 5.7 55.6 0 their base (Figure 11). The latter usually include three flowering period. In normal conditions, most of the typical male sepals and petals, six typical male stami- bisexual flowers fall (Figure 12(a)) through an nodes and three carpels. These carpels differ in size unknown physiological mechanism which is from flower to flower, depending on their position on increased by the influence of the wind that causes the rachilla. Bisexual flowers in bisexual male inflor- the friction of rachillae between them and the palms. escences are arranged conversely to those found in A limited number of these flowers develop as parthe- bisexual female date palm inflorescences – that is, nocarpic fruits composed of one, two, or three devel- inflorescence branches bear male flowers toward the oped carpels (Figure 12(b)–(e). These fruits were upper part while the bisexual flowers are found at the similar to the normal parthenocarpic fruits produced base. Interestingly, in the same inflorescence, rachil- by unpollinated flowers from female trees, only dif- lae can be composed of only male flowers or male fering from them by petal form. flowers together with bisexual flowers. Pollination After 6 months, we noticed that after self-pollination The fate of bisexual flowers produced by bisexual (Figure 13(a)), all the obtained fruits coming from female date palm bisexual flowers were parthenocarpic, except one fruit Spontaneous fate that closely resembled, in its shape, color, and struc- When the bisexual flowers enclosed by craft paper ture, a typical fruit coming from a normal cross- were allowed to develop, a large number of parthe- pollinated flower (Figure 13(b)–(d)). This fruit, pre- nocarpic (seedless) fruits were obtained; in general all sumably originating from self-fertilization, contained three carpels form fruits. These fruits were similar to a seed comparable to that of typical mature comes- the normal parthenocarpic fruits produced by unpol- tible fruit (Figure 13(e), (f) regarding volume and linated female trees. It was also observed that the mass. Nevertheless, the seed was malformed since it parthenocarpic fruits were set on typical female lacked axial symmetry and did not contain a visible inflorescences that were formed at the second embryo. This malformed seed was unable to 6 A. OTHMANI ET AL.

Ps Ps

c c St P P

S S

a b

Ps St c

p c

Figure 8. Longitudinal sections through bisexual date palm flowers (a–c) showing at once female and male organs. S: sepal, P: petal, Ps: pollen sac, C: carpel, St: stamen. Scale bar 100 µm germinate under normal conditions. It should be Discussion noted that all fruits obtained after transfer of stamens Our study demonstrates for the first time that two of bisexual flowers to the flowers of another female offshoots of a female date palm plant ‘Alligue’ pro- date palm variety ‘Deglet nour’ were parthenocarpic. duce pistillate flowers with vestigial staminodes, with a structure histologically similar to those of viable The fate of bisexual flowers produced by male staminodes from typical staminate flowers. date palms Bisexuality in date palm has been also described by Al-Beker (1972) and DeMason and Tisserat (1980)in Conventionally, whole male flowers produced during certain male date palm plants. The inability of off- the first flowering period (February to March) degen- shoot 1 to produce developed staminodes may sug- erate and eventually fall. Similar results were gest that, contrary to the environmental conditions observed with flowers produced during the first and prevailing outside the oasis, those prevailing inside the second flowering (June) of the male date palms have no influence on the genes and/or epigenetic ‘Abd1’, ’Abd2’, and ‘Abd3’. Nevertheless, some bisex- factors controlling the expression of male sexual ual flowers of the male ‘P8’, that were formed at the organs. second flowering period at the basal part of rachillae, Our results show that bisexual flowers produced developed into parthenocarpic fruits composed of by offshoots 2 and 3 of a female date palm ‘Alligue’ three non-pollinated carpels that reached maturity occupy preferentially the upper part of rachillae with after 4–5 months (Figure 14). These fruits were simi- varying numbers from year to year. Their number lar to the normal parthenocarpic fruits produced by remains lower than that of ordinary female flowers. unpollinated female trees. THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 7

n1 n2

intine

exine b c

d e

Figure 9. Longitudinal sections through pollen sac. (a) A global vision. (b–e) Structure of pollen grains. n1 and n2: generative nucleus and pollen tube nucleus. Scale bar 20 µm.

This result confirms those obtained on bisexual flow- certainly involving endogenous growth regulators ers on the rachillae of the male date palm obtained acting indirectly via genetic regulation (Chailakhyan, from the crossing Deglet Noor × L 10 BC (DeMason 1979) to reactivate the arrested staminodes in female & Tisserat, 1980). However, flowers of the male flowers and pseudocarpels in male flowers (DeMason obtained after the crossing of Hilali × 50-9 BC were & Tisserat, 1980). predominantly bisexual and produced few male flow- All bisexual flowers produced by the two studied ers (DeMason & Tisserat, 1980). date palm offshoots have three carpels that differ in The variation in the number of bisexual flowers their size which is inversely proportional to the num- from year to year can be explained by the influence of ber of staminodes that varies from one to six. Hence, the climate, especially the temperature, on the all these flowers are at once female and male, but with mechanism responsible for the induction of bisexu- different degrees of masculinization. The mechanism ality in the two studied date palms. The temperature by which a chromosomally dioecious species is acts preferentially on the upper parts of rachillae that induced or stimulated to produce hermaphrodite constitute its target sites by unknown means, but flowers remains unclear, but it certainly requires a 8 A. OTHMANI ET AL.

st p

a b

Figure 10. Longitudinal sections through bisexual date palm flower. (a) A global vision. (b) An enlarged carpel. s: sepal, p: petal, ps: pollen sac, c: carpel, st: stamen. Scale bars 100 µm (a) and 1500× (b).

female flowers, Masmoudi et al. (2009) reported that Table 3. Period of flowering in male date palm P8. addition of Indole-3-butyric acid and 6-benzylamino- Year First flowering Second flowering purine separately to MS basal medium at concentra- 2012 February June 2013 February June tions varying from 2.46 to 19.68 µM and 2.22 to 2014 February No flowering 8.88 µM, respectively, stimulates the proliferation of vestigial stamens of female flowers as in the case of number of different genetic and epigenetic factors, male flowers without blocking the carpel’s develop- from sex chromosomes to plant hormones ment, leading to morphologically typical hermaphro- (Tanurdzic & Banks, 2004). We suggest that the fac- dite flowers which are even able to produce viable tor responsible for the masculinization is likely to be pollen grains. a hormone which is normally absent or present at low Sex modification in flowers of some other dioe- concentration in ordinary female date palms, but cious plant species has also been described. For exam- produced in the two studied date palms at different ple, Komai, Shikazono, and Tanaka (2003) were able concentrations in the same inflorescence. In the lit- to produce hermaphrodite flowers from female flow- erature, many reports demonstrated an intimate cor- ers of Spinach (Spinacia oleracea L.) by using helium relation between the fluctuations of endogenous and carbon irradiation. Furthermore, sex modifica- growth regulators and sex expression in date palm. tion of flowers can be modified by using some che- For example, Leshem and Ophir (1977) found that mical treatments, such as silver thiosulfate (Ag2S2O3), female date palms contain higher endogenous gibber- which stimulated stamen development in female ellin concentrations than males. The abovementioned flowers of Silene latifolia (Law, Hardenack, & Grant, speculation is confirmed by the fact that bisexuality 2002). in date palm can be enhanced in vitro by application In oil palm (Elaeis guineensis Jacq.), Corley (1976) of exogenous growth regulators. In fact, in male demonstrated that gibberellic acid had a masculinizing flowers, DeMason and Tisserat (1980) reported that effect by privileging production of male inflorescences − the employment of 10 mg L 1 2,4-dichlorophenoxya- while reducing female ones. By studying the same cetic acid or p-chlorophenylacetic acid and 0.3% acti- plant, he postulated that ethephon and a-naphthylene vated charcoal enhanced the development and acetic acid also had an influence on the sex ratio. outgrowth of the pseudocarpels. Nevertheless, in Similarly, Komai and Masuda (2004) reported that THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 9

a b

c d

e f

Figure 11. Structure of inflorescences and flowers produced by Abd 1 and a bisexual male date palm (P8): (a) Rachillae of Abd 1. (b) Rachillae of P8 (Bf: bisexual flower at the base and Mf: male flower at the top). (c and d) Typical male flowers in frontal and top view, respectively. (e) Bisexual flower in frontal view. (f) Three kinds of carpels differing in size, produced by the male P8. Scale bar 500 µm.

Ffp

Pfb

a b

Mnf e

Pfb

Pff c d

Figure 12. The spontaneous fate of bisexual flowers of offshoots 2 and 3. (a) Fallen bisexual flowers. (b) Rachilla with bisexual and female flowers just after pollination (Pfb: parthenocarpic fruit coming from bisexual flower and Ffp: typical female flower after pollination). (c) Parthenocarpic fruits in the stage of green date. (d) Rachillae with mature and parthenocarpic fruits at the final stage of development coming from typical pollinated female flowers (Mnf: maturated normal fruit), a typical non-pollinated female flower (Pff: Parthenocarpic fruit coming from non-pollinated female flower) and a non-pollinated bisexual flower Pfb: parthenocarpic fruit coming from non-pollinated bisexual flower). (e) Four enlarged parthenocarpic fruits coming from two nonpollinated bisexual female flowers occupying the top of the rachillae). Scale bar 500 mm. 10 A. OTHMANI ET AL.

fabf b iccp

pf tf tf

fabf

pfnf pf fabf pfnbf c d

stplf sapdf saplf

stpdf e f

Figure 13. The fate of bisexual flowers produced by offshoots 2 and 3 after intervention. (a) Inflorescence covered by craft paper (iccp). (b) Fruit originated from apparent pollinated bisexual flower (fabf). (c) Fabf compared with typically fruits produced by the same plant in another inflorescence (tf) and parthenocarpic fruit (pf) produced by the same inflorescence. (d) Different kind of fruits produced by the studied plants cut longitudinally in two equal parts (tf: typical fruit, pfnf: parthenocarpic fruit originated from non-pollinated typical female flower, fabf: fruit originated from apparent pollinated bisexual flower, pfnbf: parthenocarpic fruit originated from non-pollinated bisexual flower and pf: 2 parthenocarpic fruits originated from nonpollinated bisexual flower. (e) Seeds of the apparent pollinated flower (sapfl) and the typical pollinated flower (stpf) on the lap face. (f) Seeds of the apparent pollinated flower (sapdf) and the typical pollinated flower (stpdf) on the dorsal face. Scale bars 0.05× (a), 0.1× (b), 0.2× (c), 0.25× (d) and 1× (e and f). Scale bar 500 mm. the sex expression can be modified when plants of following compatible pollination (Shivanna, spinach were regenerated from root callus by somatic Linskens, & Cresti, 1991). Pollen viability can be embryogenesis via successive generations. evaluated by direct methods, for example the induc- Examination of the longitudinal sections of appar- tion of in vitro (Sorkheh et al., 2011) and in vivo ently bisexual flowers showed morphologically nor- germination (Fakhim, Hajilou, & Zaare, 2011), and mal male and female organs. In these flowers, indirect methods using cytological parameters, as in stamens consist of a filament topped with two lobed the case of pollen staining (Abdelgadir, Johnson, & anthers. Every lobe contains four pollen sacs in which Van Staden, 2012). In our study, we did not test the spherical pollen grains are produced. The latter seem viability of pollens because stamens of bisexual flow- to be in the final ripening phase as they have two ers did not produce enough pollen grains. nuclei (generative nucleus and tube nucleus). The It is known that in spring, date palms cultivated in abovementioned result suggests that the pollen grains a desert environment flower once a year, but a second are mature but not necessary viable. To be viable, a flowering can occur in the same year in the case of an pollen grain must be able to germinate on the stigma exceptional rainfall (Johnson, 2010). Therefore, the and to deliver the sperm cells to the embryo sac second flowering is probably governed by the climate. THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 11

c d

Figure 14. The fate of bisexual flowers produced by the male date palm (P8). (a) Parthenocarpic fruits in the beginning of the stage of green date. (b) Enlarged parthenocarpic fruits after 3 months. (c) Formation of matured parthenocarpic fruits at the basal part of rachillae. (d) Enlarged matured parthenocarpic fruits. Scale bar 2 cm.

Taking into account the criteria of number of carpels with the potential to develop a large number of flowerings per year and the sex of flowers, we were parthenocarpic fruits. The authors showed that the able to demonstrate for the first time that male date structure of the expanded carpels on male plants is palm plants can be divided into three groups: (1) histologically similar to that of carpels from typical plants that produce male flowers only at the first pistillate flowers. flowering; (2) plants that produce only male flowers In higher plants, the fertilization process consists at the first flowering and at the second flowering if it in general of five steps (Lord & Russell, 2002); (1) occurs; (3) plants that produce only male flowers at pollination (the transfer of pollen from anther to the first flowering, and male and bisexual flowers at stigma) which is provided by the agencies of wind, the second flowering if it occurs. animals or water; (2) pollen germination (the emer- Following the example of bisexual inflorescences of gence of the pollen tube that transports male gametes the studied female trees, those of the bisexual male tree (sperm cells) to the egg cell in the embryo sac during (P8) are composed of two kinds, namely typical male sexual reproduction through the aperture); (3) pene- flowers that occurred generally at the upper part of tration of the pollen tube into the ovule; (4) double rachillae and bisexual flowers occurring mostly at their fertilization (one sperm fertilizes the egg in the base. Hence, the degree of feminization increases from embryo sac to produce a diploid zygote (2n) and the upper part to the basal part of rachillae, in opposi- the embryo, while the other sperm fuses with the tion to the degree of masculinization. Whether covered polar nuclei in the embryo sac to form the 3n endo- by craft paper or not, bisexual flowers failed to give sperm that provides food for the developing embryo); mature fruits with ordinary seeds; they either degen- and (5) the division of the fertilized egg (zygote). erate or give rise to parthenocarpic fruits. These obser- Consequently, fertilization failure may have multiple vations confirm the finding of DeMason and Tisserat causes. It can result from the complete absence of male (1980), who reported that in certain conditions, some organs, the failure to develop normal sporogenous tissues male plants produce bisexual flowers with expanded (no meiosis), the abortion of pollen at any step of its 12 A. OTHMANI ET AL. development, the absence of stamen dehiscence or the References inability of mature pollen to germinate on compatible Abdelgadir, H.A., Johnson, S.D., & Van Staden, J. (2012). stigma (Budar & Pelletier, 2001), or the hostility of envir- Pollen viability, pollen germination and pollen tube onmental factors (internal and external conditions, fac- growth in the biofuel seed crop Jatropha curcas tors) to pollination or germination or double fertilization. (Euphorbiaceae). South African Journal of Botany, 79, Certainly, fertilization failure depends on the 132–139. doi:10.1016/j.sajb.2011.10.005 environmental conditions and especially the tempera- Al-Beker, A.J. (1972). The date palm: A review of its past, ture that causes male sterility in many species, includ- present and the recent advances in its culture industry and trade. Baghadad: Al-Anis Press. (In Arabic). ing rice (Endo et al., 2009), canola (Brassica napus L.) Al-Dous, E.K., George, B., Al-Mahmoud, M.E., Al-Jaber, (Polowick & Sawhney, 1988) and Hordeum vulgare L. M.Y., Wang, H., Salameh, Y.M., . . . Malek, J.A. (2011). (Tadashi & Atsushi, 2008). This assumption is most De novo genome sequencing and comparative genomics likely because when covering inflorescences before of date palm (Phoenix dactylifera). Nature Biotechnology, – their anthesis by craft paper to avoid pollination by 29, 521 527. doi:10.1038/nbt.1860 Budar, F., & Pelletier, G. (2001). Male sterility in plants: other pollens, the temperature surrounding inflores- Occurrence, determinism, significance and use. Comptes cences can vary from the external temperature and rendus de l’Académie des Sciences-serie III- Sciences de la may inhibit germination of pollen grains on the stig- Vie, 6, 543–550. mas. The same result was also observed with bisexual Carpenter, J.B., & Ream, C.L. (1976). Date palm breeding, a – flowers produced by male date palm (P8). review. Date Growers Institute Report, 53,25 33. Nevertheless, when bisexual flowers were fertilized Chailakhyan, M.K.H. (1979). Genetic and hormonal regulation of growth, flowering, and sex expression in plants. by pollen from the male Abd1, they converted into American Journal of Botany, 66, 717–736. doi:10.2307/ ordinary fruits. Such a result demonstrates without 2442417 doubt that the studied date palm plant is female- Chandler, W.H. (1958). Evergreen orchards (2nd ed., pp. fertile and presumably male-sterile. 205–228). Philadelphia, PA: Lea and Fibiger. The fact of having hermaphrodite flowers in both Cherif, E., Zehdi, S., Castillo, K., Chabrillange, N., Abdoulkader, S., Pintaud, J.-C., . . . Aberlenc-Bertossi, female and male trees in date palm, a dioecious spe- F. (2013). Male-specific DNA markers provide genetic cies, suggests that date palm had bisexual or gynodioe- evidence of an XY chromosome system, a recombination cious ancestors. Also, it encourages further work to arrest and allow the tracing of paternal lineages in date develop seeded fruits after self-pollination. If we can palm. New Phytologist, 197, 409–415. doi:10.1111/ obtain complete seeded fruits after self-pollination of nph.12069 bisexual flowers of the two studied date palm plants, ChihCheng, T.C., & Robert, R.K. (2007). The date palm (Phoenix dactylifera L.): Overview of biology, uses, and we may decrease the heterozygosity of the descendants cultivation. Hortscience, 42. by decreasing chromosomal and genetic recombina- Corley, R.H.V. (1976). Sex differentiation in oil palm: tion. Such success may help in both producing self- Effects of growth regulators. Journal of Experimental progeny date palms conforming to the mother plant Botany, 27, 553–558. doi:10.1093/jxb/27.3.553 and reducing the tedious and high-cost but indispen- DeMason, D.A., & Tisserat, B. (1980). The occurrence and structure of apparently bisexual flowers in the date palm, sable manual operation of pollination in date palm. Phoenix dactylifera L. (Arecaceae). Botanical Journal of the Linnean Society, 81, 283–292. doi:10.1111/ boj.1980.81.issue-4 Endo, M., Tsuchiya, T., Hamada, K., Kawamura, S., Yano, K., Acknowledgements Ohshima, M., . . . Kawagishi-Kobayashi, M. (2009). High temperatures cause male sterility in rice plants with tran- We are grateful to Mr Yesine Rhouma for the delivery of scriptional alterations during pollen development. Plant the plant material, and to Drs Stefaan Werbrouck, Dennis Cell Physiology, 50,1911–1922. doi:10.1093/pcp/pcp135 V. Johnson, and Jameel M. Al-khayri for excellent sugges- Fakhim, R.S.H., Hajilou, J., & Zaare, N.F. (2011). Pollen tions and critically reading the manuscript. germination and pistil performance in several Iranian peach cultivars. International Journal Agriscience, 1,71–77. Johnson, D.V. (2010). Worldwide dispersal of the date palm from its homeland. Acta Horticulturae, 882, 369– Disclosure statement 375. doi:10.17660/ActaHortic.2010.882.42 No potential conflict of interest was reported by the Komai, F., & Masuda, K. (2004). Plasticity in sex expression authors. of spinach (Spinacia oleracea) regenerated from root tissues. Plant Cell Tissue Organ Culture, 78, 285–287. doi:10.1023/B:TICU.0000025665.74491.1e Komai,F.,Shikazono,N.,&Tanaka,A.(2003). Sexual mod- Funding ification of female spinach seeds (Spinacia oleracea L.) by irradiation with ion particles. Plant Cell Reports, 21, 713–717. This work was supported by the IRESA (Institution de la Law, T.F., Hardenack, S.B., & Grant, S.R. (2002). Silver Rechercheetdel’Enseignement Supérieur Agricoles)/ Centre enhances stamen development in female white campion Régional de Recherches en Agriculture Oasiennes de Degache, (Silene latifolia [Caryophyllaceae]). American Journal of Tunisia. 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