This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Environmental and Experimental Botany 71 (2011) 152–157

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

First stages of microspore reprogramming to embryogenesis through antherculture in Prunus armeniaca L.

María Antonietta Germanàa, Benedetta Chianconea, Diego Padoana, Ivett Bárányb,María-Carmen Risuenob, Pilar S. Testillanob,∗

a Dipartimento SENFIMIZO, Università degli Studi di Palermo, Viale delle Scienze 11, 90128 Palermo, Italyb Centro de Investigaciones Biológicas (CSIC), C/Ramiro del Maeztu, 28040 Madrid, Spain

a r t i c l e i n f o

Article history:Received 21 April 2010Received in revised form17 November 2010Accepted 25 November 2010

Keywords:ApricotCell structureCytochemistryMicrospore embryogenesis

a b s t r a c t

Prunus armeniaca L. is a worldwide known species, very important particularly in the Mediterraneanbasin. Microspore embryogenesis through in vitro anther culture is a widely used method to obtainhaploid and doubled haploid (DHs) plants which are being routinely used in breeding programmes fornew superior cultivar development in many crops. Haploid–diploidization through gametic embryoge-nesis allows single-step development of complete homozygous lines from heterozygous parents. In thecase of fruit crops, with long reproductive cycle, a high degree of heterozygosity, large size, and, often,self-incompatibility, there is no way to obtain haploidization through conventional methods. Inductionof microspore embryogenesis in vitro is switched by a stress treatment. In many species, heat or coldstress has been reported to trigger pollen embryogenesis, the response being genotype dependent. In thepresent work we analyzed whether microspore reprogramming could be induced in apricot cultivars bycold stress through anther culture. We report the development of an in vitro anther culture protocol inP. armeniaca L. and analyse the response of several cultivars to stress treatments and culture media forinducing pollen embryogenesis. Results showed the formation of multicellular pollen and proembryos.The effect of two culture media in the embryogenic response was also analyzed, being the responsesgenotype-dependent. Monitoring of the cellular changes on the microspores was performed by struc-tural and confocal microscopy analyses. Results indicated that the reprogramming of the microspore andthe first steps of the embryogenic pathway have been achieved in different varieties of P. armeniaca,which constitutes a crucial step in the design of protocols for the regeneration of microspore-derivedembryos and DH plants, for future potential applications in breeding programmes of this economicallyimportant fruit tree.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Among the biotechnological tools that can greatly help fruitcrop breeding, haploid and doubled haploid production, throughgametic embryogenesis, allows the single-step development ofcomplete homozygous lines from the heterozygous parents,increasing the efficiency of perennial crop breeding programmes.Haploid plants are sporophytes carrying the gametic chromosomenumber (n instead of 2n), and doubled haploids (DHs) are haploidsthat underwent, spontaneous or induced, chromosome duplica-tion. Haploid and DHs have a potential use in mutation research,selection, genetic analysis, transformation and in the production ofhomozygous cultivars also required to utilize as parental lines forF1 hybrids (heterosis). Fruit crops are characterized by the high

∗ Corresponding author.E-mail address: testillano@cib.csic.es (P.S. Testillano).

heterozygosity of the genomes, the long duration of generationcycle with a long juvenile period, the large size, and, often, theself-incompatibility, and, for these reasons, there is no way to pro-duce homozygous breeding lines through conventional methodsthat involve several generations of selfing. Considerable researchhas been carried out since the 1970s to obtain haploids for fruit treebreeding, through gametic embryogenesis, but, not always, theywere successful (Ochatt and Zhang, 1996; Maluszynski et al., 2003;Germanà 2006; Srivastava and Chaturvedi, 2008).

Haploids can be mainly induced by regeneration from the malegamete (pollen embryogenesis) or from the female gamete (gyno-genesis), and, particularly, in vitro anther or isolated microsporecultures are the most effective and widely used methods of pro-ducing haploids and DHs. Haploid production from Datura innoxiaanther in vitro cultured was first reported by Guha and Maheshwari(1964), since then pollen embryogenesis has been reported formore than 250 species, belonging to 100 genera and more than40 families, including Cruciferae and Gramineae [1], while many

0098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.envexpbot.2010.11.011

Author's personal copy

M.A. Germanà et al. / Environmental and Experimental Botany 71 (2011) 152–157 153

Fig. 1. Correlation between phenological stages of flower buds and pollen development. Sequential stages of floral development in apricot (a–c) and microspore developmentin vivo (d–i). Toluidine blue staining (d–f) and DAPI staining for DNA under confocal microscope (g–i). (d and g) Young vacuolated microspore; (e and h) late vacuolatedmicrospore; (f and i) bicellular pollen. Thin arrows: generative cell; thick arrows: vegetative cell. Bars represent: 10 �m.

members of Leguminosae family and many woody plants are ratherrecalcitrant and only in a few of them efficient in vitro protocolsto induce pollen embryogenesis have been reported (Bueno et al.,1997, 2005; Germanà, 2006, 2009; Höfer et al., 1999). Particularly,haploid plant regeneration has not been achieved from in vitroculture of anther or isolated microspores in temperate stone fruitspecies (Peixe et al., 2004).

Prunus armeniaca L. is a worldwide known species with over3.8 million tonnes of fruits produced in 2009 (FAOSTAT Database),very important particularly in the Mediterranean basin. Apricot isdiploid with a monoploid number of chromosomes n = x = 8. Apri-cot has been primarily domesticated in China, but has a secondarycentre of origin in Middle East (Watkins, 1976). It is a traditionalfruit of North Africa and, in Algeria, it is considered a very prof-itable fruit crop for the local climate. Evidence of its cultivationin Maghreb can be traced as far as the XII century. Fruits are con-sumed fresh or dried and are considered as a staple food crop by thelocal populations (Panaud et al., 2002). Limited in vitro culture pro-tocols for organ regeneration and propagation have been reportedfor Prunus species (Espinosa et al., 2006; Koubouris and Vasilakakis,2006; Arbeloa et al., 2009; Canli and Tian, 2009; Petri and Scorza,2010) Regarding research on pollen embryogenesis in apricot, Harnand Kim (1972) obtained callus formation from apricot anther cul-ture, but its ploidy level was not determined. The formation of callifrom cultured anthers of apricot ‘Harcot’, as well as the differenti-ation of nodular structures have been also reported by Peixe et al.(2004). The authors studied the influence of the temperature dur-ing the pre-treatment and evaluated, by flow cytometry, the ploidylevel of calluses that ranged from haploid to octoploid.

Induction of microspore embryogenesis in vitro is switched bya stress treatment, in many cases temperature stress. In differentherbaceous and tree species, heat or cold stress has been reported totrigger pollen embryogenesis, the response being genotype depen-

dent (reviewed in Maluszynski et al., 2003). We hypothesized thatmicrospore reprogramming could be induced in apricot cultivarsby cold stress through anther culture. In the present study, weanalysed whether a cold stress treatment followed by the in vitroculture in appropriate culture media was able to induce embryo-genic responses in the microspores of several apricot cultivars;and whether the response was genotype dependent. The resultsshowed the formation of multicellular pollen and proembryos indifferent cultivars of P. armeniaca through the reported in vitroanther culture protocol, confirming the hypothesis. The first stagesof microspore reprogramming were analyzed also through struc-tural and confocal microscopy observations, performed at differenttimes of the culture, to monitor the main cellular changes of theapricot microspores during the anther culture.

2. Materials and methods

2.1. Plant material and anther culture

One year branches were harvested in February from a col-lection field, located in Lascari (Palermo, Italy) and, as inductivepre-treatment, they were subjected to 4 ◦C, in the dark, for twoweeks. In order to evaluate the influence of genotype on thegametic embryogenesis induction from microspores, five culti-vars of P. armeniaca L., and, more in details, three cultivars fromItaly: Ninfa, Palumella, Portici, one from Canada: Orange Red, andone from Tunisia: Sajeb, were tested. After the cold inductivepre-treatment, the branches were forced under 25 ◦C day/nighttemperature and 16-h photoperiod until the flower buds reachedthe phenological stage described by Fleckinger (1964) in whichbuds swell get longer and petals appear (Fig. 1a–c). Particularly,at this phase, inside the anthers there are mostly microspores atthe vacuolated stage, that is the most suitable for gametic embryo-

Author's personal copy

154 M.A. Germanà et al. / Environmental and Experimental Botany 71 (2011) 152–157

genesis induction (González-Melendi et al., 1995; Peixe et al.,2004). To check the pollen developmental stage, one anther pereach flower size was selected and the microspores were stainedby 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, SIGMA)solution (1 mg ml−1) and then observed under a fluorescent micro-scope. Only flower buds of the same size of the ones containingvacuolate microspores (Fig. 1d and e) were selected for antherculture.

Flower buds, collected from branches, were sterilized by immer-sion for 3 min in 70% ethanol, followed by immersion in sodiumhypochlorite solution (about 1.5% active chlorine in water) con-taining a few drops of Tween 20, and finally rinsed three times for3 min with sterile distilled water. About 50 anthers were placedin each Petri dish, containing 10 ml of solid medium. Anthers ofall genotypes were cultured on medium P, reported by Germanàand Chiancone (2003). To test influence of the another mediumcomposition on apricot gametic embryogenesis, anthers of threeselected genotypes (Ninfa, Palumella, Sajeb), were placed on themedium NN4, N6 of Chu (1978), added with 4% sucrose, 4.52 �M2,4-dichlorophenoxyacetic acid (2,4-D); 2.85 �M indole-3-aceticacid (IAA) and 4.56 �M zeatin, resulted the best one in the experi-ments carried out by Peixe et al. (2004). Calli obtained from antherculture were transferred to a regeneration medium containinghalf basal MS medium (Murashige and Skoog, 1962) added with4% sucrose and 5.70 �M IAA, 4.55 �M zeatin and 4.55 �M thidi-azuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea: TDZ), as reported byPeixe et al. (2004). 10 Petri dishes were prepared per each geno-type and per each medium (500 anthers per each genotype and pereach medium). Petri dishes were sealed with parafilm, incubatedat 26 ± 1 ◦C, in the dark for the first 30 days and then placed undercool white fluorescent lamps (Philips TLM 30W/84, France) with aphotosynthetic photon flux density of 35 �mol m−2 s−1 and a pho-toperiod of 16 light hours. The number of anthers not developed,swollen, or with calli was observed after two months of culture ineach Petri dish. These values were used to calculate means. Differ-ences among cultivars were tested by analysis of variance at P < 0.01level. Differences among means were tested by Tukey multiplecomparison test.

2.2. Cellular architecture

2.2.1. Fixation and processing for light microscopyIn order to characterize the main changes in the cellular

architecture of microspores in culture, as well as to identify thedevelopmental stages of the formation of microspore-derived mul-ticellular structures, a microscopical analysis was carried out.Samples, at different times: just collected from the tree, afterone week, two weeks and two months of culture were selected,processed, and resin embedded. Particularly, anthers were fixedin 4% paraformaldehyde in phosphate buffered saline (PBS), pH7.3, overnight at 4 ◦C. After washing in PBS, samples were dehy-drated through an acetone series, infiltrated and embedded inTechnovit 8100 acrylic resin (Kulzer, Germany), at 4 ◦C, as previ-ously described (Solís et al., 2008) except for the time of infiltrationwhich was increased in 48 h for apricot anthers, to optimize thepenetration of the resin into the tissue. Semithin sections werestained with different methods and analyzed under light and con-focal laser scanning microscopy.

2.2.2. Toluidine blue staining and iodide–potassium-iodide (I2KI)cytochemistry for starch

Toluidine blue staining was used to observe the cellular orga-nization under light microscopy. Iodide-based cytochemistry wasperformed at specific stages for localization of starch granules.Staining solutions (0.075% toluidine blue in water; 2 g of KI and0.2 g of I in 100 ml of water) were applied on Technovit sec-

tions for 10–15 min. After rinsing and drying, preparations weremounted in Eukitt and observed under bright field in a LeitzLaborlux 12 microscope, equipped with a DP10 Olympus digitalcamera.

2.2.3. DAPI staining for nuclei observation on resin sections andconfocal analysis

DAPI staining was analyzed under confocal microscopy for thenuclei examination. 1 �g/ml 4′,6-diamidino-2-phenylindole dihy-drochloride (DAPI) solution in PBS was applied for 15 min tosemithin Technovit sections, which after rinsing were mountedin Mowiol and observed in a confocal laser scanning microscopeLeica CLSM TCS SP2 under UV irradiation, DAPI fluorescence opticalsections and their projections were captured.

3. Results and discussion

3.1. Anther culture progression revealed genotype-dependentresponses to embryogenesis-inductive conditions (stress andmedium)

An analysis to correlate the phenological stage of flower buds,anther size and pollen developmental stage was firstly carried outin order to establish a convenient selection criterion of the buds.Several flower bud stages containing anthers of different sizeswere determined according to the total length and their sepal-petallengths ratio. DAPI staining for DNA was performed on anthersto reveal the developmental stage of the microspores containedin each bud/anther type. Selected stages showed young and latevacuolated microspores (Fig. 1d, e, g and h) inside buds whichexhibited the petals appearing with a length lesser than the sepals(Fig. 1a and b), and bicellular pollen (Fig. 1f and i) and mature polleninside the longer buds, with the length of petals higher than thesepals (Fig. 1c). These results permitted us to select the buds atthe most appropriate stage for the in vitro culture, those containingvacuolate microspores (Fig. 1a and b), according to most reportson pollen embryogenesis (González-Melendi et al., 1995; Ramírezet al., 2003; Peixe et al., 2004; Germanà et al., 2006; Solís et al.,2008).

Different features have been described in anther cultures assigns of initiation of a morphogenic response and the change of thedevelopmental pathway. After one week of culture, while the non-responsive anthers decreased in volume and turgenscence (Fig. 2b),in other anthers, the swelling, that is the first anatomical changeaccompanying the morphogenic response (Germanà et al., 2006),was observed (Fig. 2c) and keep swelling for more than four weeks;then, some of the swollen anthers started to produce callus (Fig. 2dand e). Different types of callus were observed: in few cases calliwere white, transparent and friable (Fig. 2d), but in most cases, calliwere yellowish (Fig. 2e), with some globular structures (Fig. 2f).DAPI staining performed after two months of culture, on antherswith globular structures, showed some multinucleated structures(Fig. 2g), indicating that the microspores were induced to followthe sporophytic pathway.

Different responses were observed among anthers cultured onmedium P (Table 1). Statistical analysis showed that the best resultswere obtained from the cultivar Sajeb, Palumella and Ninfa; infact, these cultivars showed the lowest percentage of not devel-oped anthers (41.8%, 49,4% and 57,9% respectively) and the highestpercentages of swollen anthers (56.3%, 49,8% and 41,4% respec-tively). No statistically significant differences were observed amongcultivars regarding callus production (Table 1). These three cul-tivars were selected for the culture in the medium NN4, whichwas previously reported for anther culture of another cultivarof apricot (Peixe et al., 2004). The cultivar Ninfa gave the best

Author's personal copy

M.A. Germanà et al. / Environmental and Experimental Botany 71 (2011) 152–157 155

Fig. 2. Progression of the in vitro anther culture in apricot. (a) Vacuolate microspore at the initiation of in vitro culture, DAPI staining; (b) not developed anther after onemonth in culture, cv. Sajeb; (c) swollen anther after one month in culture, cv. Sajeb; (d) anther with white callus; (e) anther with yellow callus; (f) anther with globularstructures, cv. Ninfa; (g) multinucleated microspore, DAPI staining, cv. Sajeb. Bars represent in (a and g): 10 �m.

Table 1Response of five apricot cultivars to the induction medium P.

Cultivar Not developedanthers %

Swollenanthers %

Anthers withcallus %

Ninfa 57.9 ab 41.4 bc 0.8Orange Red 70.5 a 28.3 c 1.2Palumella 49.4 bc 49.8 abc 0.8Portici 59.2 ab 39.8 c 1.0Sajeb 41.8 c 56.3 a 1.8

Values within each column followed by different letters are significantly differentat P ≤ 0.01 (Tukey’s test).

results, showing the highest percentage of anthers producingcallus (12.2%) (Table 2), a percentage much higher than that reg-istered in the medium P (0.8%) (Table 1). Moreover, there was astatistically significant interaction between the two factors (geno-type and medium). The best medium for apricot anther culturewas the NN4; in fact, for Ninfa as well as for Sajeb, the low-est percentage of not developed anthers (respectively 40.5% and33.9%) and the statistical highest percentage of anthers with cal-lus (12.2% and 6.2%) were observed in this medium (Table 2).Probably, P medium, set up for citrus anther culture and charac-terized by a higher number of components (amino acid, coconutwater, ascorbic acid, etc.) and by a lower concentration of growthregulators is less suitable for apricot pollen embryogenesis. Thequantitative study also showed that the best response was foundin Ninfa cultivar since it displayed a notably higher percent-age of anthers with callus (12.2%), in comparison with the othercultivars.

Table 2Effect of the two induction media on the in vitro anther development.

Cultivar Medium Not developedanthers %

Swollenanthers %

Anthers withcallus %

Ninfa P 57.9 41.4 0.7NN4 40.5 47.3 12.2

Palumella P 49.4 49.8 0.8NN4 53.1 45.1 1.8

Sajeb P 41.8 56.3 1.8NN4 33.9 59.8 6.3

Cultivara P ≤ 0.001 P ≤ 0.001 P ≤ 0.001Mediuma P ≤ 0.001 P = 0.339 P ≤ 0.001Cultivar × mediuma P ≤ 0.001 P = 0.024 P ≤ 0.001

a P values for main factors and interactions of two-way ANOVA (Tukey’s test).

3.2. Cellular architecture analysis showed the formation ofmulticellular pollen and embryos

The progression of the in vitro anther cultures was monitoredand the changes in the cellular organization of the microsporeswere analyzed in comparison with the gametophytic development.At the beginning of the culture (Fig. 3a), the microspores exhib-ited the typical architecture of the vacuolated microspores duringthe gametophytic development (Fig. 1e and h). After one week,microspores developed in culture, after embryogenesis inductiveconditions, exhibited differential features, showing two nuclei withsimilar size and organization (Fig. 3b and c), in contrast with thetwo different nuclei of the bicellular pollen developed in vivo (com-pare Fig. 1f with 3b). These two-cell structures indicated that themicrospore in vitro underwent a symmetrical division and switchedfrom their gametophytic developmental pathway towards prolif-eration and were the result of the first embryogenic division ofthe microspore which still exhibited large cytoplasmic vacuoles(Fig. 3b). At later stages, cytoplasmic vacuoles progressively dis-appeared and multicellular structures were observed with densecytoplasms, still surrounded by exine, the pollen wall (Fig. 3d) Theiodide-based cytochemistry revealed the presence of starch gran-ules in the multicellular structures (data not shown), as it wasreported in the microspore-derived multicellular proembryos ofother woody species like Citrus ssp. (Ramírez et al., 2003) and Ery-obotrya japonica Lindl. (Germanà et al., 2006).

At later stages, after two months in culture, the responsiveanthers were broken and showed callus and globular structures(Fig. 3e). The microscopical analysis of sections of these brokenanthers revealed remnants of the somatic tissues of the anther androunded undifferentiated cells with large vacuoles and differentsizes, typical callus cells (Fig. 3f), which appeared grouped and dis-persed inside and out of the anther. Interestingly, at the interior ofthe broken anthers rounded multicellular masses, similar to multi-cellular proembryos, were found (Fig. 3e and f). They were formedby cells with a typical architecture of proliferative cells with largenuclei, dense cytoplasms and thin walls (Fig. 3f and g); the globularproembryos appeared as individual structures limited by a layer ofcells completely different, with elongated shape and cytoplasmicinclusions (Fig. 3f and g). The presence of specific layers of cellsand cell wall components surrounding multicellular organogenicmasses and proembryos has been reported in other organogenicin vitro systems (Fortes et al., 2002; Bárány et al., 2010b). It hasbeen suggested that these peripheral components were specific

Author's personal copy

156 M.A. Germanà et al. / Environmental and Experimental Botany 71 (2011) 152–157

Fig. 3. Cellular monitoring during in vitro anther culture. Microspore development (a–d) and proembryos formation (e–h). Toluidine blue staining (a, b, d–g), DAPI stainingfor DNA under confocal microscope (c), and iodide-staining for starch (h). (a) Vacuolate microspore at the beginning of the culture; (b and c) microspore-derived two-cellstructures after one week of culture; (d) multicellular pollen grain with dense cytoplasms after two weeks of culture; (e) panoramic view of an anther after 2 months inculture showing a rounded multicellular mass or proembryo (arrow); (f) proembryo surrounded by callus cells; (g) detail of proembryo cells; (h) starch cytochemistry, theproembryo cells (in the centre) show small and scarce starch granules whereas the surrounding cells exhibit larger and more abundant starch inclusions. Bars represent in(a–d): 10 �m, in (e, f and h): 100 �m, in (g): 50 �m.

of the periphery of cellular masses with organogenic competence(Fortes et al., 2002). These proembryos would be originated by thesubsequent proliferation of the multicellular pollen grains afterthe exine breakdown, as it has been found in other anther cul-tures for embryogenesis induction of woody species (Ramírez et al.,2003, 2004). The iodide-based cytochemistry for starch revealedvery small starch grains in the cytoplasm of the proembryo cellswhereas the cells surrounding the proembryos and the rest ofthe somatic cells displayed very large starch inclusions (Fig. 3h).A lower amount or absence of intracellular starch granules havebeen described in young microspore-derived proembryos of otherspecies, associated with cell proliferation (Bárány et al., 2005; Solíset al., 2008), the increase in the starch accumulation being relatedwith differentiation events (Bárány et al., 2005,2010a).

The evolution of the in vitro system described here, fromtwo-cell and multicellular pollen grains to large multicellularproembryos indicated that the reprogramming of the microspore

and the first steps of the embryogenic pathway have been achievedby the protocol assayed here in different varieties of P. armeniaca.

4. Conclusions

Breeding of perennial plants is usually cumbersome and time-consuming due to their long reproductive cycle, high degree ofheterozygosity and complex reproductive biology and, so, thepotential of gamete biotechnology can represent, particularly forthem, a great advantage in comparison with the conventionalmethods. In the present paper, a new anther culture system hasbeen developed for microspore embryogenesis in apricot. The evo-lution of the in vitro system has been characterized from two-celland multicellular pollen grains to large multicellular proembryos,indicating that the reprogramming of the microspore and the firststages of the embryogenic pathway have been achieved. Thesefindings constitute a crucial step in the design of protocols for

Author's personal copy

M.A. Germanà et al. / Environmental and Experimental Botany 71 (2011) 152–157 157

the regeneration of microspore-derived embryos and DH plants,very important for future potential applications in breeding pro-grammes of this fruit tree of economic interest.

Many are the factors affecting the success of the pollen embryo-genesis (microspore developmental stage, anther or floral budpre-treatments, medium composition, culture conditions, etc.) and,for this reason, a deeper knowledge and understanding of thisprocess and how the factors drive morphogenic competence anddevelopment, will enable the effective deployment of gameticembryogenesis and haploid technology in the improvement of apri-cot and generally of fruit trees. Further efforts are necessary toobtain regeneration of microspore-derived embryos and plantlets.

Acknowledgements

This work is a collaboration between the CSIC (Spain) and theUniversita’ degli Studi di Palermo (Italy) in the frame of the ItalianCORI 2006 and it is also a collaboration between the CNR (Italy) andthe CSIC in the frame of the Spanish-Italian Joint Project CSIC-CNR2008IT0046. This work was partially supported by Spanish MICINNprojects BFU2008-00203 and AGL2008-04255.

References

Arbeloa, A., Daorden, M.E., García, E., Andreu, P., Marín, J.A., 2009. In vitro culture of“Myrolaban” (Prunus cerasifera Ehrh.) embryos. Hortscience 44, 1672–1674.

Bárány, I., Fadón, B., Risueno, M.C., Testillano, P.S., 2010a. Microspore reprogram-ming to embryogenesis induces changes in cell wall and starch accumulationdynamics associated with proliferation and differentiation events. Plant Sig-nalling and Behavior 5, 341–345.

Bárány, I., Fadón, B., Risueno, M.C., Testillano, P.S., 2010b. Cell wall componentsand pectin esterification levels as markers of proliferation and differentiationevents during pollen development and embryogenesis. J. Exp. Bot. 61, 1159–1175.

Bárány, I., Gonzalez-Melendi, P., Mityko, J., Fadon, B., Risueno, M.C., Testillano, P.S.,2005. Microspore-derived embryogenesis in Capsicum annuum L.: subcellularrearrangements through development. Biol. Cell 97, 709–722.

Bueno, M.A., Gómez, A., Boscaiu, M., Manzanera, J.A., Vicente, O., 1997. Stress inducedhaploid plant production from anther cultures of Quercus suber. Physiol. Plant.99, 335–341.

Bueno, M.A., Pintos, B., Höfer, M., Martin, A., 2005. Pro-embryos induction from Oleaeuropaea L. isolated microspore culture. Acta Physiol. Plant. 27, 695–701.

FAOSTAT Database http://faostat.fao.org/default.aspx.Canli, F.A., Tian, L., 2009. Regeneration of adventitious shoots from mature stored

cotyledons of Japanese plum (Prunus salicina Lind1). Sci. Hortic. 120, 64–69.

Chu, C., 1978. The N6 medium and its applications to anther culture of cereal crops.In: Proc. Symp. Plant Tissue Cult ,. Science Press, Peking, pp. 43–50.

Espinosa, A.C., Pijut, P.M., Michler, C.H., 2006. Adventitious shoot regeneration androoting of Prunus serotina in vitro cultures. Hortscience 41, 193–201.

Fleckinger, J., 1964. In: Grisvard, P., Chaudun, V. (Eds.), Le bon jardinier: La MaisonRustique, Paris., pp. 362–372.

Fortes, A.M., Testillano, P.S., Risueno, M.C., Pais, M.S., 2002. Studies on callose andcutin during the expression of competence and determination for organogenicnodule formation from internodes of Humulus lupulus var. Nugget. Physiol. Plant.116, 113–120.

Germanà, M.A., 2006. Doubled haploid production in fruit crops. Plant Cell Tiss. Org.Cult. 86, 131–146.

Germanà, M.A., 2009. Haploid and doubled haploids in fruit trees. In: Touraev, A.,Forster, B., Jain, M. (Eds.), Advances in Haploid Production in Higher Plants.Springer, pp. 241–263.

Germanà, M.A., Chiancone, B., 2003. Improvement of the anther culture protocol inCitrus clementina Hort. ex Tan. Plant Cell Rep. 22, 181–187.

Germanà, M.A., Chiancone, B., Levy-Guarda, N., Testillano, P.S., Risueno, M.C., 2006.Development of multicellular pollen of Eriobotrya japonica Lindl. through antherculture. Plant Sci. 171, 718–725.

González-Melendi, P., Testillano, P.S., Ahmadian, P., Fadon, B., Vicente, O., Risueno,M.C., 1995. In situ characterization of the late vacuolate microspore as a conve-nient stage to induce embryogenesis in Capsicum. Protoplasma 187, 60–71.

Guha, S., Maheshwari, S.C., 1964. In vitro production of embryos from anthers ofDatura. Nature 204, 497.

Harn, C., Kim, M.Z., 1972. Induction of callus from anthers of Prunus armeniaca.Korean J. Breed. 4, 49–53.

Höfer, M., Touraev, A., Heberle-Bors, E., 1999. Induction of embryogenesis fromisolated apple microspores. Plant Cell Rep. 18, 1012–1017.

Koubouris, G., Vasilakakis, M., 2006. Improvement of in vitro propagation of apricotcultivar “Bebecou”. Plant Cell Tiss. Org. Cult. 85, 173–180.

Maluszynski, M., Kasha, K.J., Forster, B.F., Szarejko, I. (Eds.), 2003. Doubled HaploidProduction in Crop Plants, A Manual. Kluwer Academic Publishers, Dordrecht,The Netherlands.

Murashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassayswith tobacco tissue cultures. Physiol Plant. 15, 473–497.

Ochatt, S.J., Zhang, Y.X., 1996. In vitro haploidization of fruit trees. In: Jain,S., Sopory, S.K., Veilleux, R.E. (Eds.), In Vitro Haploid Production in HigherPlants, vol. 3. Kluwer Academic Publisher, Dordrecht, The Netherlands,pp. 193–210.

Panaud, O., Chaib, A., Sarr, A., 2002. Dynamic conservation of apricot Prunus arme-niaca in saharian oases: use of AFLP markers to assess genetic diversity intraditional orchards. Euphytica 128, 301–305.

Peixe, A., Barroso, J., Potes, A., Pais, M.S., 2004. Induction of haploid morphogeniccalluses from in vitro cultured anthers of Prunus armeniaca cv. ‘Harcot’. PlantCell Tiss. Org. Cult. 77, 35–41.

Petri, C., Scorza, R., 2010. Factors affecting adventitious regeneration from in vitroleaf explants of “Improved French” plum, the most important dried plum cultivarin the USA. Ann. Appl. Biol. 156, 79–89.

Ramírez, C., Chiancone, B., Testillano, P.S., García-Fojeda, B., Germanà, M.A., Risueno,M.C., 2003. First embryogenic stages of Citrus microspore-derived embryos. ActaBiol. Cracov. Bot. 45, 53–58.

Ramírez, C., Testillano, P.S., Pintos, B., Moreno, M.A., Bueno, M.A., Risueno, M.C.,2004. Changes in pectins and MAPKs related to cell development duringearly microspore embryogenesis in Quercus suber L. Eur. J. Cell Biol. 83,213–225.

Solís, M.T., Pintos, B., Prado, M.J., Bueno, M.A., Raska, I., Risueno, M.C., Testillano, P.S.,2008. Early markers of in vitro microspore reprogramming to embryogenesis inolive (Olea europaea L.). Plant Sci. 174, 597–605.

Srivastava, P., Chaturvedi, R., 2008. In vitro androgenesis in tree species: an updateand prospect for further research. Biotech. Adv. 26, 482–491.

Watkins, R.R., 1976. Cherry, plum, peach, apricot and almond. In: Simmonds, N.W.(Ed.), Prunus spp. (Rosaceae). Evolution of Crop Plants. Longman, London, pp.242–247.