Istituto di Biologia e Biotecnologia Agraria IBBA, C.N.R., UO di Pisa, via Moruzzi 1, Pisa, Italy. 2

CARYOLOGIA Vol. 64, no. 2: 223-234, 2011 Cytogenetic and histological approach for early detection of mantled somaclonal variants of oil palm regenerated by somatic embryogenesis: first results on the characterization of regeneration system GIORGETTI LUCIA 1, *, MONICA RUFFINI CASTIGLIONE 2, ALESSANDRA TURRINI 3, VITTORIA NUTI RONCHI 1 and CHIARA GERI 1 1 Istituto di Biologia e Biotecnologia Agraria IBBA, C.N.R., UO di Pisa, via Moruzzi 1, Pisa, Italy. 2 Dipartimento di Biologia, Università di Pisa, Via Ghini 5, Pisa, Italy. 3 Dipartimento di Biologia delle Piante Agrarie, Università di Pisa, Viale delle Piagge 23, Pisa, Italy. Abstract The occurrence of anomalous cytological events during the cell proliferation of plant primary explants, and the prevalence of polyploidization and chromosome reduction events, mainly induced by in vitro culture stress, generate large genome variability among cultured committed cells and successively regenerated plants. A thorough cytological analysis on different embryogenic and non-embryogenic oil palm (Elaeis guineensis Jacq.) calli was performed, with the aim to identifying chromosome instability as a possible cause of somaclonal variants in the regenerated plants. Besides, the process of embryoids formation from embryogenic calli was dissected, setting up a fast and reliable system of tissue culture from zygotic embryos. Our results demonstrated that in the cultured explants of oil palm, similarly to other plant species, the initial events leading to embryogenic/regeneration commitment were managed by a reprogramming of somatic cells towards a gametelike state, including chromosomes segregation and the occurrence of haploid gamete-like cells. The intrinsic instability of oil palm chromosomes was also verified. Key words: Cytogenetic anomalies; Oil palm; Organogenesis; Somaclonal variation; Somatic embryogenesis. INTRODUCTION Oil palm Elaeis guineensis Jacq. is the first world source of vegetable oil used mainly in alimentary field (high quality oil and its derivates) and in industrial activity (biofuel) (SUMATHI et al. 2008; USDA 2008). Oil palm is a monocotyledon plant with a single meristematic apex. Since the most cultivated oil palms are F1 hybrids (dura x pisifera = tenera), giving highly variable yields, huge efforts have been exerted since 1970s to establish in vitro propagation protocols, in order to obtain the most productive elite plants via somatic embryogenesis (JALANI et al. 1997). Embryogenic cultures were obtained starting from *Corresponding author: Phone +39 050 3153089; Fax +39 050 315 3328; e-mail l.giorgetti@ibba.cnr.it somatic explants of different origin such as leaf, root, immature inflorescence etc. Commonly, for large scale in vitro propagation, immature leaves of selected plants were preferred for their best convenience, but the percentage of in vitro induction is still very low (1-3% of leaf explants can produce embryogenic callus) and the relative time quite unpredictable (1-6 months, till more than one year) as reported by DE TOUCHET et al. 1991; DUVAL et al. 1995; WONG et al. 1997. Nowadays, although tissue culture procedures allow a massive clonal propagation of oil palm plants, little is known about the cause of the somaclonal variation shown by the regenerants and affecting particularly floral organs. In fact, while regenerants showing vegetative abnormalities are immediately eliminated, the ones with abnormal floral organs (mantled phenotype, i.e. feminization of flower male parts, which can result in complete sterility in the most severe cases) can be individuated only at the

224 GIORGETTI, RUFFINI CASTIGLIONE, TURRINI, NUTI RONCHI and GERI reproductive age (5-6 years old plants), resulting in great crop losses (CORLEY et al. 1986). By means of cytogenetic and molecular approaches, attempts were carried out in order to early verify (in vitro diagnosis) floral abnormalities, but few hint can be proposed to solve the problem (RI- VAL et al. 1998a, 1998b; JALIGOT et al. 2002). In addition it was observed that, also in oil palm, somaclonal variation can be minimized depending on the hormonal source used for cell culture induction (THAWARO and TE-CHATO 2009). More recently a 20 years survey on oil palm cultures maintained on a free phytohormone medium showed that embryogenetic capacity was not lost with the passing of time, even if morphogenic quality of the lines and plant survival rate after planting out decreased; interestingly, mantled phenotype did not seem to be related to culture ageing (KONAN et al. 2010). This work proposes a cytological screening of a large number of oil palm cultures having different origin, age and embryogenic capability, obtained from leaf explants, in order to describe the possible mechanisms leading to abnormal variants, such as the mantled phenotype in the regenerated plants. In particular the chromosome variability increasing during oil palm in vitro culture was described taking into account previous observations of NUTI RONCHI s team on chromosome reducing and polyploidization mechanisms operating in other plant systems (NUTI RONCHI et al. 1992a, 1992b). These mechanisms, widely demonstrated, are briefly reported in the following paragraphs. Chromosome reducing mechanisms: a) Prophase chromosome reduction, which separates chromosomes into two or more groups, according to ploidy, during different prophase stages (NUTI RONCHI and TERZI 1988; NUTI RONCHI et al. 1992a). As outcome prophase-like nuclei are situated close to each other in the same cell with chromosomes being directly unthreaded into prophase configurations; b) Reduction grouping mechanisms: in this case, nuclei are split, after prophase, directly in numerous arrested metaphase chromosomes arranged in groups in the same cell (NUTI RONCHI et al. 1992a); c) Somatic meiosis: the term somatic meiosis is used to describe divisions of somatic plant cells in culture that mimic the process of chromosome segregation and reduction normally occurring in the reproductive organs (NUTI RONCHI et al. 1992b; NUTI RONCHI 1995). This mechanism may produce gamete-like cells as in normal meiosis (GIORGETTI et al. 1995). Polyploidization-inducing mechanisms: a) C- metaphase: Colchicines-like metaphases lead to mitosis abortion and formation of a restitution polyploid nucleus. This process can lead directly to aneuploidy when one or more chromosome are lost in the process; b) Chromosome endoreduplication: this mechanism generates polyploidy in plants, both in vivo and in vitro, through DNA endoreduplication; endoreduplicated cells can be distinguished from polyploids of other origins during the first mitosis due to the presence of diplochromosomes or quadruplochromosomes (i.e. 4 or 8 chromatids held together by relational coiling in prophase or at the centromere in metaphase). The presence of 2,4-Dichlorophenoxyacetic acid (2,4-D), as the hormonal component of the medium, has been often considered the principal agent responsible for this and other chromosomal abnormalities, but other factors, as temperature variation or physical-chemical stresses, may contribute to polyploidization through such a mechanism (NUTI RONCHI 1990). Moreover our previous results from other plant species demonstrated that proliferating primary explants invariably undergo processes mimicking sexual maturation, included somatic meiosis and floral-like primordia differentiation (GIORGETTI et al. 1995; GERI et al. 1999; PITTO et al. 2001). These events occur in somatic cultured cells very early in vitro, and their outcome is due to a genetic reprogramming that, while erasing the previous information related to the somatic differentiated status, directs the committed cells to a gamete-like state (totipotency), prerequisite for embryogenesis or regeneration. We could demonstrate in oil palm culture also the emergence of the phenomena described above. Since the callus-induction system from immature leaf is too unpredictable to be used routinely in the dissection of the very early critical events in vitro, the culture of zygotic embryos turned out to be the most reliable. This approach made possible a cyto-histological analysis along the first 14 days of culture, using as control zygotic embryos on hormone-free medium. MATERIALS AND METHODS Culture conditions, cytological and histological analysis of oil palm calli - Oil palm calli were obtained from immature leaves in three different Malaysian laboratories: Malaysian Palm Oil Board (MPOB, Persiaran Institusi, Bandar Baru

DETECTION OF MANTLED SOMACLONAL VARIANTS OF OIL PALM 225 Bangi, 43000 Kajang, Selangor, Malaysia), Applied Agricultural Resources (AAR, 47000 Sg Buloh Selangor, Malaysia), and Federal Land Development Authority (FELDA, Jalan Gurney Satu, 54000 Kuala Lumpur, Malaysia). The research material derived from Eleis guineensis Jacq. hybrids DxP = var tenera (Deli x Yangambi i.e. Asia x Central Africa-Congo). A total of 28 calli were analysed: 4 embryogenic calli from MPOB (obtained from immature leaves of DxP hybrid, plant 3/GP13); 10 embryogenic calli and 10 non-embryogenic calli from AAR (both obtained from immature leaves of DxP, plant A220 and A221); 4 embryogenic calli from FELDA (obtained from immature leaves DxP hybrid, plant FC). Oil palm calli were maintained in MS medium (MURASHIGE and SKOOG 1962) added with 2,4-D 1mg/L and gelrite 1%, and monthly sub-cultured. Embryos formation was induced by the removal of hormone in the MS medium. In AAR non-embryogenic calli, no somatic embryos were produced in hormone free medium as cells continued to proliferate as undifferentiated calli. For cytogenetic analyses oil palm calli were fixed in Carnoy fixative (ethanol-acetic acid 3:1 v/v) overnight and processed according to Feulgen s method: 1 hour hydrolysis in HCl 5 N at room temperature followed by 45 minutes staining with Schiff s Reagent (BDH) (GIORGETTI et al. 1995). Stained samples were placed on few drops of 45% acetic acid and squashed for cytological analysis. For each callus not less than 650 mitotic cells were scored. For histological analysis oil palm samples, collected during in vitro culture and regeneration process, were fixed in ethanol-acetic acid (3:1, v/v), dehydrated in an alcohol series, cleared in xylene, and embedded in paraffin. 12 μm thick sections were cut in a rotary microtome, stained with Feulgen s method and counterstained with haematoxylin (Activity 2, Shandon), (PITTO et al. 2001). Fig. 1 Cytological analysis of different oil palm in vitro samples showing abnormal mitotic divisions; (a, b, c, d) reduction grouping during prophase generating chromosome diminution; (e) pro-metaphase and (f) c-metaphases with 32 chromosomes; (g) polyploidy c-metaphase; (h) polyploid metaphase arranged in multiple mitotic spindles; (i) mitosis with evident homologous chromosome pairing; (j) reduction grouping at late prophase and metaphase; (k) chromosome bridge at anaphase and (l) chromosome lagging at metaphase generating aneuploidy.

226 GIORGETTI, RUFFINI CASTIGLIONE, TURRINI, NUTI RONCHI and GERI Cytological and histological analysis during the first days of zygotic embryo culture - Zygotic embryos aseptically excised from dry seeds (DxP 309/125 genotype from MPOB plantation) were grown on solid (agar) MS medium supplemented with 2,4D 1mg/L (MS+), and as control in hormone free MS medium (MS-). To determine the localization and the typology of the early divisions, the cultured embryos were collected during the first 14 days and fixed in Carnoy. This material was stained following Feulgen s procedure and squashed for cytological analysis (GIORGETTI et al. 1995) or wax included for sectioning (PITTO et al. 2001). Root tips were used as controls. The frequency of mitotic anomalies was estimated through the analysis of a minimum 650 mitotic cells per sample. Data Analysis - Data from individual assays correspond to the mean values ± standard error (S.E.). Statistically significant differences among the groups were identified using ANOVA and Bonferroni post hoc test, with values of p<0.01 sufficient to reject the null hypothesis (STOLINE et. 1981). RESULTS Cytological analysis of oil palm calli - Initially a detailed cytological analysis was performed on the different embryogenic and non-embryogenic oil palm calli, coming from different companies and sent us by MPOB: four embryogenic calli (first group) from FELDA, four embryogenic calli (second group) from MPOB, ten embryogenic calli (third group), and ten non-embryogenic calli (fourth group) from AAR. All tested calli derived from Eleis guineensis Jacq. var tenera and were initially obtained from young leaves in the three different Malaysian Laboratories. Subsequently all the oil palm cultures were subcultured in the same conditions in our laboratory. A large chromosomal instability was identified within each group. We could evidence chromosome reducing mechanisms, including pairing and somatic meiosis, polyploidization-inducing mechanisms and chromosome aberrations. Figure 1 shows some representative examples of these cytological abnormalities. In figure 1a, b, Fig. 2 Cytological analyses after Feulgen s staining of a) FELDA calli (4 calli, group 1) (open bars), b) MPOB calli (4 calli, group 2) (light grey bars), c) AAR embryogenic calli (10 calli, group 3) (dark grey bars), d) AAR non-embryogenic calli (10 calli, group 4) (black bars). The data were collected in a single histogram for every group. Normal mitoses are represented in class 1, polyploidization mechanisms, including C-metaphases, endoreduplications, polyploid metaphases, are represented in class 2; reduction mechanisms, including prophase reduction, reduction grouping, haploid metaphases, are shown in class 3; chromosome aberrations, consisting of chromosome lagging and chromosome bridges, are shown in class 4. For each callus at least 660 mitosis were analysed. ** = ANOVA significant at p<0.01; NS = not significant. Results with the same letters are not significantly different from each other by Bonferroni s multiple comparison test. = ± standard error.

DETECTION OF MANTLED SOMACLONAL VARIANTS OF OIL PALM 227 c, d, the mechanism of reduction grouping during prophase is shown: chromosomes are split into two or more groups at prophase giving origin to chromosome reduction in daughter cells. Pro-metaphases with countable chromosomes (Fig. 1e) and c-metaphases (Fig. 1f, c-metaphase with 32 chromosomes, Elaeis guineensis Jacq. 2n number, and Fig. 1g, polyploid c-metaphase) were also evidenced. C-metaphase anomalies indicated a defect in the polar spindle formation and successively the failure of the proper mitotic division giving origin to polyploid or aneuploid cells. In other cases multiple mitotic spindles were formed in large polyploid metaphases (Fig. 1h). Further cytological abnormal mechanisms as homologous chromosome pairing at prometaphase (Fig. 1i) and reduction grouping at late prophase and metaphase (Fig. 1j) were observed. Moreover, aberrations generating aneuploidy, such as chromosome bridge at anaphase and chromosome lagging at metaphase were detected (Fig. 1k, l). To quantify the occurrence of the different cytological mechanisms generating genome variability in cultured oil palm, a thorough analysis of the mitotic features was carried out. Since the frequencies of the above mentioned mechanisms within each group were statistically analogous (p < 0.01, ANOVA and Bonferroni post hoc test), the data were collected in a single histogram for every group (Fig. 2). Moreover the different cytological mechanisms were gathered in four classes: 1 normal mitosis; 2 polyploidization mechanisms including C-metaphases, endoreduplications and polyploid metaphases; 3 reduction mechanisms including prophase reduction, reduction grouping and haploid metaphases; 4 chromosome aberrations consisting of chromosome lagging and chromosome bridges. The cytological analysis showed the following results: polyploidization mechanisms (class 2) were 11.24% of total mitoses in FELDA calli, 18.78% in MPOB calli, 25.95% in AAR embryogenic calli and 19.56% in AAR non embryogenic calli. One-way ANOVA and Bonferroni s multiple comparison test showed significant differences only between group 1 (FELDA calli) and group 3 (AAR embryogenic calli). Re- Fig. 3 Cytological analyses of oil palm root apices obtained from germinated seeds (309/125 genotype) (white bars) and the in vitro regenerated plant A220-255 (black bars). Normal mitoses are represented in class 1, polyploidization mechanisms, including C-metaphases, endoreduplications, polyploid metaphases, are represented in class 2; reduction mechanisms, including prophase reduction, reduction grouping, haploid metaphases, are shown in class 3; chromosome aberrations, consisting of chromosome lagging and chromosome bridges, detected in oil palm calli (fig. 2) are not present in root apices (class 4). A minimum of 660 mitotic cells was scored. ** = ANOVA significant at p< 0.01; NS = not significant. Results with the same letters are not significantly different from each other by Bonferroni s multiple comparison test. = ± standard error.

228 GIORGETTI, RUFFINI CASTIGLIONE, TURRINI, NUTI RONCHI and GERI duction mechanisms were generally more represented in all groups: reduction mitoses were 37.38% of total mitoses in FELDA calli, 33.8% in MPOB calli, 24.15% in AAR embryogenic calli and 20.95% in AAR non-embryogenic calli. One-way ANOVA and Bonferroni multiple comparison tests showed significant differences in FELDA calli versus AAR embryogenic calli and AAR non-embryogenic calli. Furthermore similar statistically significant differences were found in MPOB calli versus AAR embryogenic calli and AAR non-embryogenic calli. Chromosome aberrations were present at low levels, 3% of total mitoses in FELDA calli, 1.91% in MPOB calli, 0.39% in AAR embryogenic calli and 1% in AAR non-embryogenic calli. Cytological analysis of seedling root meristems - Since the genetic variability of the mother plant play a considerable role in the fate of the cloned offspring, the same analyses were performed in Fig. 4 Differentiation process in embryogenic oil palm in vitro cultures from embryogenic nodular calli: (a) embryogenic masses, different phases of shoots formation, (b, c, d, e), till plantlets stage (f).

DETECTION OF MANTLED SOMACLONAL VARIANTS OF OIL PALM 229 vivo on root tips of seedlings (309/125 genotype from MPOB plantation) as controls, as well as on the root tip meristems of regenerated plants. Unexpectedly the same instability of the chromosomal asset, to a lesser extent, was demonstrated in both systems (Fig. 3). In fact, polyploidization and reduction mechanisms were present, showing for seedling root tips abnormalities around 9.6% and 4.4% respectively on total analysed mitoses. These percentages were significantly higher in the regenerated plant root tips from A220#255 AAR calli (20.2% and 24.60 % respectively). Chromosome aberrations were absent both in control as in regenerated plant apices (Fig. 3). These data show that oil palm cells are particularly prone to genome instability and that in vitro culture conditions enhance the intrinsic characteristic of this plant genetic background. Histological analysis of embryogenic calli - The histological analysis was performed on some embryogenic calli with the aim to track embryo formation and to examine the relative position of embryogenic cells. For this purpose the process of embryo formation was dissected on embryogenic calli, maintained in culture in our lab in MS+ medium and transferred in MS- to induce embryos as described in literature. A preliminary analysis was performed at the stereomicroscope and the most interesting calli were sectioned after paraffin inclusion. The study on calli revealed the presence of organised structures (Fig. 4 a, b), which, when sectioned, appeared as meristematic newly formed centres (Fig. 5a-c), and successively gave origin to embryoids. Besides, the modalities of somatic embryo formation from embryogenic calli emerged more as an organogenesis process than a true embryonic one (Fig. 4 c-f). All em- Fig. 5 Histological analysis of embryogenic calli showed in Fig. 4 during the first differentiation steps leading to pro-embryogenic masses formation (a, b, c), embryogenic layer arrangement giving origin to shoots formation (d, e); arrow indicates the epidermal layer of the in vitro regenerated plantlets (e).

230 GIORGETTI, RUFFINI CASTIGLIONE, TURRINI, NUTI RONCHI and GERI bryogenic calli transferred in MS- solid medium developed meristematic centres resembling shoot formation deep into the callus (Fig. 5a-c). Next to the meristematic zones newly formed vascular bundles appeared and successively the shoots emerged on the callus surface. They remained connected to the inner callus tissue until an epidermal layer was formed to separate the plantlet from each other and from the nearby undifferentiated cells (Fig. 5 d, e). Callus induction from oil palm zygotic embryos - The standard protocol of callus induction, from very young leaves, is the only one accessible for the companies, based on a huge number of explants, with a very low level of callogenesis and even less of regeneration. But the unpredictability of this process compelled us to establish, only for research purposes, a different callogenesis protocol, starting from zygotic embryos (Fig. 6 a). This protocol resulted a fast and reliable one, having as control the germinating seeds, put in parallel with the dedifferentiating ones, in the hormone free medium. On this material the leading biological events along the commitment process were traced down. Cyto-histological analyses put in evidence that, in the embryos cultured in MS+ medium, the bulk of mitotic activity started from the day 5 th even if occasionally divisions occurred at day 3 rd and 4 th. On the contrary no mitoses were found in MS- cultured embryos until the day 6 th as reported in figure 7. The first divisions were localised in the procambial zone of the haustorium and in the embryonic apices (Fig. 6 b, c). After 20 days of in vitro culture in MS+ medium, the haustorium part developed characteristic finger-like structures (Fig. 6 d) resembling young male inflorescences (Fig. 6 e). The cytological analysis of mitotic patterns along the dedifferentiation process of cultured zygotic embryos in MS+ medium, till the 14 th day (Fig. 7), confirmed the presence of anomalous divisions. Polyploidization mechanisms (class 2) were present at the highest percentage during the first days of culture as reported in figure 7 (day 5 th 24.22%; day 6 th 28.11% and day 8 th 26,04%), but had a great relevance also in zygotic embryos in hormone free medium (day 6 th 26% and day 14 th 16,51%). Moreover reduction mechanisms (class 3) as well were detectable in MS+ cultured zygotic embryos at higher percentage since the day 5 th (day 5 th 29.60%, day 6 th 39.27%, day 8 th 30.3% and day 14 th 42.05%), differing from the rate found in control zygotic embryos cultured in MS- (day 6 th 27.48%, day 8 th 24.78% and day 14 th 17.77%). Statistical analysis evidenced the significance of the differences between zygotic embryos cultivated in MS+ and MS- (as control) with some exceptions (Fig. 7). This result is a further demonstration of the hormone effect enhancing the genomic imbalance of oil palm cells in vitro. DISCUSSION The obtained data demonstrate that oil palm species possess a natural instability of the chromosomal and genomic asset. Even the root tips of control plantlets (germinated from seeds) show some abnormalities, suggesting that they are structurally present in the mother plant. These abnormalities are more frequent during in vitro culture, not only in the callus but also in the regenerating structures. Such a large unsteadiness is present in all the analyzed clones, from the same or from different sources. One-way ANOVA and Bonferroni multiple comparison test show differences in FELDA calli versus AAR embryogenic calli and AAR nonembryogenic calli respectively. Differences are significant also in MPOB calli versus AAR embryogenic calli and, interestingly, between AAR non-embryogenic calli and AAR embryogenic calli (same genetic background). This result is interesting, since it is the only one significant between AAR embryogenic and non-embryogenic calli: it could imply that aberrations in particular are strongly selected against in the acquisition of embryogenic competence. Although with statistically significant differences all the four groups of calli present the same phenomena of genome variability, that are irrespective of the age of the culture, or the embryonic potential. A possible implication could be that the detected differences are correlated with the different manipulation in vitro. In fact the cultures coming from AAR mainly show different rates of cytological anomalies if compared to the other materials, probably due to the different protocol of culture induction and/or manipulation. As it is clear from the data, most of the abnormalities can be recognised as events leading to reduction or increase of the chromosomal asset. But, whereas the last incident may not affect too much growth and development if the chromosomal number is properly balanced, the reducing mechanism can produce cells with altered genome size (aneuploids, triploids etc.) or cells with segregated chromosomes, i.e. recessive genes may reappear. Particularly interesting is the presence of chro-

DETECTION OF MANTLED SOMACLONAL VARIANTS OF OIL PALM 231 mosomal aberrations, quite frequent in some cases. The chromosomal bridges (indicating a chromosomal break followed by reunion), the chromosomal fragments, the lagging chromosomes (likely lost during the division), are all symptoms of severe genomic imbalance. It is worth noting, with regard to the influence of the abnormal divisions and genomic aberrations on the regeneration ability, that, even extreme, chromosomal variability does not always interfere with the regeneration capacity and with the initial growth and differentiation, at Fig. 6 Oil palm Zygotic embryo (309/125 genotype) in vitro cultivated in MS medium added with 1mg/L 2,4-D. (a) whole embryo excised from seed in which the root (R) the shoot (S) and the haustorium (H) vascular bundles are well evident at stereomicroscope. (b, c) histological sections of zygotic embryo at day 5 th of culture showing the occurrence of the first divisions mainly localised in the procambial zone of the haustorium (b) and in the embryonic apices (c). (a, b, c) Feulgen s staining. (d) zygotic embryo after 20 days of in vitro culture; it is worth noting how haustorium part developed characteristic finger-like structures resembling young male inflorescences primordia showed in (e).

232 GIORGETTI, RUFFINI CASTIGLIONE, TURRINI, NUTI RONCHI and GERI least up to the plantlet stage. But further growth is strongly prevented, apparently only normal chromosomal numbers allowing the regenerated plantlets to growth till maturity (NUTI RONCHI et al. 1981). On the other hand, it is a common observation that only few oil palm plantlets grow to maturity out of hundred of regenerated plantlets (malformed or arrested in growth), which are constantly discharged. The results also confirm that the oil palm embryos produced according to the companies protocols are not true somatic embryos, but appear as shoots, originated from a group of few or more cells (SCHWENDIMAN et al. 1988). This does not exclude that in some cases true somatic embryos could be produced (TE-CHATO and HILAE 2007). Moreover, besides the intrinsic oil palm genetic instability, the length of the leaf culture is undoubtedly a strong factor promoting every kind of genetic anomalies, since it is well known that a partial genomic reprogramming occurs at every sub culture (GIORGETTI et al. 1995; GERI et al. 1999), generating abnormalities accretion. On the other hand recent observations have highlighted that oil palm cultures maintained on a free phytohormone medium over a very long period, have not shown any clear and systematic increase in the abnormality rate with the age of in vitro culture (KONAN et al. 2010). Even if leaf culture is a well established protocol assuring the propagation of elite plants and recently refined leading to less variants than before, it is not very efficient if compared to the protocols based on zygotic embryo culture. The histological analysis of this system, after having tracked the anatomical development of induced somatic embryos, can imply the single cell origin of oil palm plants in vitro regenerated as previously suggested (KANCHANAPOOM and DOMYOAS 1999). Being the callus production from zygotic embryos significantly faster than from leaf explants, it was possible to study the early events occurring during in vitro dedifferentiation of somatic tissue towards callogenesis. The data obtained during the first days of zygotic embryos cultures confirmed the presence of cytological Fig. 7 Cytological analysis of excised zygotic embryos 309/125 of oil palm during the first 14 days of in vitro culture (T5, T6, T8, T14= 5, 6, 8, 14 days of culture respectively) in MS+ medium (white bars) and during germination in MS- medium (black bars). Class 1 corresponds to Normal mitoses; class 2 consists in Polyploidization mechanisms (C-metaphases, endoreduplications, endomitosis, polyploidy metaphases); class 3 reduction mechanisms (prophase reduction, reduction grouping, haploid metaphases); class 4 corresponds to chromosome aberrations includes chromosome lagging and chromosome bridges. For each sample a minimum of 1000 mitotic cells was scored. ** = ANOVA significant at p< 0.01; NS = not significant. Results with the same letters are not significantly different from each other by Bonferroni s multiple comparison test. I = standard error.

DETECTION OF MANTLED SOMACLONAL VARIANTS OF OIL PALM 233 mechanisms leading to genome variability and particularly the occurrence of chromosome reduction (prophase reduction and reduction grouping) already found during the characterisation of oil palm calli derived from leaf explants. These data are in accordance with our previous study performed in other plant species. It was demonstrated that a fundamental prerequisite to obtaining embryogenic cultures and somatic embryos consists in a reprogramming of somatic cells towards gamete-like conditions (totipotency acquisition) during the in vitro culture (GIORGETTI et al. 1995; PITTO et al. 2001). Moreover our study suggests that the oil palm explants are able to differentiate structures resembling inflorescences primordia. By means of in situ hybridization experiments with floral oil palm probes provided by MPOB, it was documented the expression of floral specific genes in the reproductive-like structure differentiated in explants in vitro (GIORGETTI et al. 2007b). In addition, microdensitometric analyses showed a decrease of the DNA content in the regenerated plants born from embryogenic calli formed by zygotic embryos in culture (GIOR- GETTI et al. 2007a). These data are in accordance also with those on the model system of carrot somatic embryogenesis, where it was demonstrated the modulation of DNA content per nucleus along the regeneration process (GERI et al. 1999). Our results so far obtained all point towards the same conclusion: oil palm system is an intrinsically variable one, and this variability is greatly enhanced by the tissue culture. Moreover the process leading to callus formation and embryogenic acquisition in oil palm follows closely the same pattern already demonstrated for other plant species. Particularly the steps related to chromosomes reduction, competent gamete-like cells production and DNA modulation (GIOR- GETTI et al. 1995, 2007a), all of them introduce an effective somaclonal variation chance in the offspring. The possibility to analyse in the same manner the mantled variant, genetic and/or epigenetic, should be strongly pursued: it can help to elucidate at which stage the abnormal phenotype has more probabilities to arise, and even to establish a predictive test. Acknowledgments This work was supported by grants from Malaysian Palm Oil Board (MPOB), Ministry of Primary Industries Malaysia (Contract research project CBR-96-004). The experiments in this study were performed in accordance with the current laws of Italy. 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