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Histological comparison between wheat embryos developing in vitro from isolated zygotes and those developing in vivo

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Abstract

There is currently great interest shown in understanding the process of embryogenesis and, due to the relative inaccessibility of these structures in planta, extended studies are carried out in various in vitro systems. The culture of isolated zygotes in particular provides an excellent platform to study the process of in planta embryogenesis. However, very few comparisons have been made between zygotic embryos grown entirely in cultures and those grown in vivo. The present study analyses the differences and similarities between the in vitro and in vivo development of wheat zygotic embryos at the level of morphology and histology. The study was possible thanks to an efficient culture system and an appropriate method of preparing isolated wheat zygotes for microscopy. The in vitro embryos were fixed, embedded and sectioned in the two-celled, globular, club-shaped and fully differentiated stages. Embryos developing in vitro closely followed the morphology of their in planta counterparts and their cell types and tissues were also similar, demonstrating the applicability of the present culture system for studying the process of zygotic embryogenesis. However, some important differences were also detected in the case of in vitro development: the disturbance of or lack of initial polarity led to changes in the division symmetry of the zygotes and subsequently to the formation of uniform cells in the globular structures. Presumably, differences between the in vitro and in planta environments resulted in a lower level of differentiation and maturation in in vitro embryos and in abundant starch and protein accumulation in the scutellum.

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Abbreviations

CBB:

Coomassie Brilliant Blue R250

CFW:

Calcofluor white

DAP:

Days after pollination

DAPI:

4′,6-Diamidino-2-phenylindole

ELS:

Embryo-like structure

HAP:

Hours after pollination

PAS:

Periodic acid-Schiff

RAM:

Root apical meristem

SAM:

Shoot apical meristem

References

  • Alemanno L, Berthouly M, Michaux-Ferriere N (1997) A comparison between Theobroma cacao L. zygotic embryogenesis and somatic embryogenesis from floral explants. In Vitro Cell Dev Biol Plant 33:163–172

    Article  Google Scholar 

  • Bakos F (2007) In vitro embryo development in gametophytic cell lines of cereal species. Ph.D thesis, Faculty of Science, Eötvös Loránd University, Budapest

  • Bakos F, Darkó É, Pónya Z, Barnabás B (2003a) Regeneration of fertile wheat (Triticum aestivum L.) plants from isolated zygotes using wheat microspore culture as nurse cells. Plant Cell Tissue Organ Cult 74:243–247

    Article  CAS  Google Scholar 

  • Bakos F, Darkó É, Pónya Z, Barnabás B (2003b) Application of wheat (Triticum aestivum L.) microspore culture and ovaries to raise wheat zygotes in vitro. Acta Biol Crac Bot 45:107–110

    Google Scholar 

  • Bakos F, Jäger K, Barnabás B (2005) Regeneration of haploid plants after distant pollination of wheat via zygote rescue. Acta Biol Crac Bot 47:167–171

    Google Scholar 

  • Batygina TB (1969) On the possibility of separation of a new type of embryogenesis in angiosperms. Rev Cytol Biol Veg 32:335–341

    Google Scholar 

  • Batygina TB (1997) Embryogenesis in Gramineae. In: Batygina TB (ed) Embryology of flowering plants, terminology and concepts, vol 2. World and Family, St. Petersburg, pp 528–538

    Google Scholar 

  • Belmonte MF, Stasolla C (2007) Applications of dl-buthionine-[S, R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca) somatic embryo development. Plant Cell Rep 26:517–523

    Article  PubMed  CAS  Google Scholar 

  • Bennett MD, Rao MK, Smith JB, Bayliss MW (1973) Cell development in the anther, the ovule, and the young seed of Triticum aestivum L. var. Chinese Spring. Philos Trans R Soc London Ser B 226:39–81

    Article  Google Scholar 

  • Bennett MD, Smith JB, Barclay I (1975) Early seed development in the Triticeae. Philos Trans R Soc London Ser B 272:199–227

    Article  Google Scholar 

  • Belmonte MF, Ambrose SJ, Ross AR S, Abrams SR, Stasolla C (2006) Improved development of microspore derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism. Physiol Plant 127:690–700

    Article  CAS  Google Scholar 

  • Bonet FJ, Olmedilla A (2000) Structural changes during early embryogenesis in wheat pollen. Protoplasma 211:94–102

    Article  Google Scholar 

  • Cawood AH, Potter U, Dickinson HG (1978) An evaluation of Coomassie blue as a stain for quantitative microdensitometry of protein in section. J Histochem Biochem 26:645–650

    CAS  Google Scholar 

  • Emons AMC, Kieft H (1991) Histological comparison of single somatic embryos of maize from suspension cultures with somatic embryos attached to callus cells. Plant Cell Rep 10:485–488

    Article  Google Scholar 

  • Emons AMC, de Does H (1993) Origin and development of embryo and bud primordia during maturation of embryogenic calli of Zea mays. Can J Bot 71:1349–1356

    Article  Google Scholar 

  • Emons AMC, Samallodroppers A, Vandertoorn C (1993) The influence of sucrose, mannitol, l-proline, absicisic acid and gibberellic acid on the maturation of somatic embryos of Zea mays L from suspension cultures. J Plant Physiol 142:597–604

    CAS  Google Scholar 

  • Faure JE, Mogensen HL, Kranz E, Digonnet C, Dumas C (1992) Ultrastructural characterization and three-dimensional reconstruction of isolated maize (Zea mays L.) egg cell protoplasts. Protoplasma 171:97–103

    Article  Google Scholar 

  • Fehér A, Pasternak TP, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tissue Organ Cult 74:201–228

    Article  Google Scholar 

  • Felföldi K, Purnhauser L (1992) Induction of regenerating callus cultures from immature embryos of 44 wheat and 3 triticale cultivars. Cereal Res Comm 20:273–277

    Google Scholar 

  • Fernandez S, Michaux-Ferriere N, Coumans M (1999) The embryogenic response of immature embryo cultures of durum wheat (Triticum durum Desf.): histology and improvement by AgNO3. Plant Growth Regul 28:147–155

    Article  CAS  Google Scholar 

  • Ferrie AMR, Keller WA (2007) Optimization of methods for using polyethylene glycol as a non-permeating osmoticum for the induction of microspore embryogenesis in the Brassicaceae. In Vitro Cell Dev Biol 43:348–355

    Article  CAS  Google Scholar 

  • Fransz PF, Schel JHN (1991) An ultrastructural study on the early development of Zea mays somatic embryos. Can J Bot 69:858–865

    Article  Google Scholar 

  • Fransz PF, Schel JHN (1994) Ultrastructural studies on callus development and somatic embryogenesis in Zea mays L. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry. (Maize vol 25). Springer, New York, pp 50–65

    Google Scholar 

  • Gaj MD (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. Plant Growth Regul 43:27–47

    Article  CAS  Google Scholar 

  • Godbole S, Sood A, Sharma M, Nagar PK, Ahuja PS (2004) Starch deposition and amylase accumulation during somatic embryogenesis in bamboo (Dendrocalamus hamiltonii). J Plant physiol 161:245–248

    Article  PubMed  CAS  Google Scholar 

  • Hause B, Vanveenendaal WLH, Hause G, van Lammeren AAM (1994) Expression of polarity during early development of microspore-derived and zygotic embryos of Brassica napus L. cv topas. Bot Acta 107:407–415

    Google Scholar 

  • He YC, Sun MX, Yang HY (2004) Regeneration of fertile plants from isolated tobacco zygotes by in vitro culture. Chin Sci Bull 49:810–814

    Google Scholar 

  • He YC, He YQ, Tang M, Sun MX (2006) An improved method for the preservation and location of single cells during the embedding procedure for transmission electron microscopy. S Afr J Bot 72:298–301

    Article  Google Scholar 

  • He YC, He YQ, Sun MX, Yang HY (2007) Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation. Plant J 49:515–527

    Article  PubMed  CAS  Google Scholar 

  • Herman EB (ed) (2008) Media and Techniques for Growth, Regeneration and Storage: 2005–2008. Volume 12 of “Recent Advances in Plant Tissue Culture”. Agricell Report, Shrub Oak

  • Holm PB, Knudsen S, Mouritzen P, Negri D, Olsen FL, Roué C (1994) Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilized egg cell. Plant Cell 6:531–543

    Article  PubMed  CAS  Google Scholar 

  • Hoshino Y, Scholten S, von Wiegen P, Lörz H, Kranz E (2004) Fertilization-induced changes in the microtubular architecture of the maize egg cell and zygote—an immunocytochemical approach adapted to single cells. Sex Plant Reprod 17:89–95

    Article  CAS  Google Scholar 

  • Indrianto A, Barinova I, Touraev A, Heberle-Bors E (2001) Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo. Planta 212:163–174

    Article  PubMed  CAS  Google Scholar 

  • Jane WN (1999) Ultrastructure of embryo development in Arundo formosana Hack. (Poaceae). Int J Plant Sci 160:46–63

    Article  Google Scholar 

  • Jones TJ, Rost TL (1989a) Histochemistry and ultrastructure of rice (Oryza sativa) zygotic embryogenesis. Am J Bot 76:504–520

    Article  Google Scholar 

  • Jones TJ, Rost TL (1989b) The developmental anatomy and ultrastructure of somatic embryos from rice (Oryza sativa L.) scutellum epithelial cells. Bot Gaz 150:41–49

    Article  Google Scholar 

  • Kärkonen A (2000) Anatomical study of zygotic and somatic embryos of Tilia cordata. Plant Cell Tissue Organ Cult 61:205–214

    Article  Google Scholar 

  • Kovács M, Barnabás B, Kranz E (1995) Electro-fused isolated wheat (Triticum aestivum L.) gametes develop into multicellular structures. Plant Cell Rep 15:178–180

    Article  Google Scholar 

  • Kovács M, Barnabás B, Kranz E (1994) The isolation of viable egg cells of wheat (Triticum aestivum L.). Sex Plant Reprod 7:311–312

    Article  Google Scholar 

  • Kranz E, Lörz H (1993) In vitro fertilisation with isolated, single gametes results in zygotic embryogenesis and fertile maize plants. Plant Cell 5:739–746

    Article  PubMed  Google Scholar 

  • Kranz E, Kumlehn J (1999) Angiosperm fertilisation, embryo and endosperm development in vitro. Plant Sci 142:183–197

    Article  CAS  Google Scholar 

  • Kranz E, Scholten S (2008) In vitro fertilization: analysis of early post-fertilization development using cytological and molecular techniques. Sex Plant Reprod 21:67–77

    Article  Google Scholar 

  • Kumlehn J, Lörz H, Kranz E (1998) Differentiation of isolated wheat zygotes into embryos and normal plants. Planta 205:327–333

    Article  CAS  Google Scholar 

  • Kumlehn J, Lörz H, Kranz E (1999) Monitoring individual development of isolated wheat zygotes: a novel approach to study early embryogenesis. Protoplasma 208:156–162

    Article  Google Scholar 

  • Kumlehn J, Kirik V, Czihal A, Altschmied L, Matzk F, Lörz H, Bäumlein H (2001) Parthenogenetic egg cells of wheat: cellular and molecular studies. Sex Plant Reprod 14:239–243

    Article  Google Scholar 

  • Kurczynska EU, Gaj MD, Ujczak A, Mazur E (2007) Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh. Planta 226:619–628

    Article  PubMed  CAS  Google Scholar 

  • Le Q, Gutierrez-Marcos JF, Costa LM, Meyer S, Dickinson HG, Lörz H, Kranz E, Scholten S (2005) Construction and screening of subtracted cDNA libraries from limited populations of plant cells: a comparative analysis of gene expression between maize egg cells and central cells. Plant J 44:167–178

    Article  PubMed  CAS  Google Scholar 

  • Leduc N, Matthys-Rochon E, Rougier M, Mogensen L, Holm PB, Magnard JL, Dumas C (1996) Isolated maize zygotes mimic in vivo embryonic development and express microinjected genes when cultured in vitro. Dev Biol 177:190–203

    Article  PubMed  CAS  Google Scholar 

  • Maraschin SF, de Priester W, Spaink HP, Wang M (2005) Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. J Exp Bot 56:1711–1726

    Article  PubMed  CAS  Google Scholar 

  • McManus JCF (1948) Histological and histochemical uses of periodic acid. Stain Technol 23:99–108

    PubMed  CAS  Google Scholar 

  • Naumova TN, Matzk F (1998) Differences in the initiation of the zygotic and parthenogenetic pathway in the Salmon lines of wheat: ultrastructural studies. Sex Plant Reprod 11:121–130

    Article  Google Scholar 

  • Nawy T, Lukowitz W, Bayer M (2008) Talk global, act local—patterning the Arabidopsis embryo. Curr Opin Plant Biol 11:28–33

    Article  PubMed  CAS  Google Scholar 

  • Nickle TC, Yeung EC (1993) Failure to establish a functional shoot meristem may be a cause of conversion failure in somatic embryos of Daucus carota (Apiaceae). Am J Bot 80:1284–1291

    Article  Google Scholar 

  • Ning J, Peng XB, Qu LH, Xin HP, Yan TT, Sun M (2006) Differential gene expression in egg cells and zygotes suggests that the transcriptome is restructed before the first zygotic division in tobacco. FEBS Lett 580:1747–1752

    Article  PubMed  CAS  Google Scholar 

  • Norstog K (1972) Early development of barley embryo fine structure. Am J Bot 59:123–132

    Article  Google Scholar 

  • Okamoto T, Higuchi K, Shinkawa T, Isobe T, Lörz H, Koshiba T, Kranz E (2004) Identification of major proteins in maize egg cells. Plant Cell Physiol 45:1406–1412

    Article  PubMed  CAS  Google Scholar 

  • Okamoto T, Kranz E (2005) In vitro fertilization—a tool to dissect cell specification from a higher plant zygote. Curr Sci 89:1861–1869

    CAS  Google Scholar 

  • Okamoto T, Scholten S, Lörz H, Kranz E (2005) Identification of genes that are up- or down-regulated in the apical or basal cell of maize two-celled embryos and monitoring their expression during zygote development by a cell manipulation- and PCR-based approach. Plant Cell Physiol 46:332–338

    Article  PubMed  CAS  Google Scholar 

  • Pretová A, Obert B, Bartosová Z (2006) Haploid formation in maize, barley, flax, and potato. Protoplasma 228:107–114

    Article  Google Scholar 

  • Pulido A, Bakos F, Castillo A, Vallés MP, Barnabás B, Olmedilla A (2005) Comparison of cytological and ultrastrucutral changes induced in anther and isolated-microspore cultures in barley: presence of Fe deposits in isolated-microspore cultures. J Struct Biol 149:170–181

    Article  PubMed  CAS  Google Scholar 

  • Pulido A, Hernando A, Bakos F, Méndez E, Devic M, Barnabás B, Olmedilla A (2006) Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development. J Exp Bot 57:2837–2846

    Article  PubMed  CAS  Google Scholar 

  • Raghavan V, Olmedilla A (1989) Spatial patterns of histone mRNA expression during grain development and germination in rice. Cell Differ Dev 27:183–196

    Article  PubMed  CAS  Google Scholar 

  • Randolph LF (1936) Developmental morphology of the caryopsis in maize. J Agric Res 53:881–916

    Google Scholar 

  • Rani V, Raina SN (2000) Genetic fidelity of organized meristem-derived micropropagated plants: a critical reappraisal. In Vitro Cell Dev Biol Plant 36:319–330

    Article  CAS  Google Scholar 

  • Schel JHN, Kieft H (1986) An ultrastructural study of embryo and endosperm development during in vitro culture of maize ovaries (Zea mays). Can J Bot 64:2227–2238

    Article  Google Scholar 

  • Seguí-Simarro JM, Nuez F (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore derived embryogenesis. Physiol Plant. doi:10.1111/j.1399-3054.2008.01113.x

  • Shayakhmetov IF, Shakirova FM (1996) Somatic embryogenesis in wheat cell suspension cultures in the presence of abscisic acid. Russian J Plant physiol 43:88–90

    CAS  Google Scholar 

  • Smart MG, O’Brien TP (1983) The development of the wheat embryo in relation to the neighboring tissues. Protoplasma 114:1–13

    Article  Google Scholar 

  • Sprunck S, Baumann U, Edwards K, Langridge P, Dresselhaus T (2005) The transcript composition of egg cells changes significantly following fertilization in wheat (Triticum aestivum L.). Plant J 41:660–672

    Article  PubMed  CAS  Google Scholar 

  • Steffen JG, Kang IH, Macfarlane J, Drews GN (2007) Identification of genes expressed in the Arabidopsis female gametophyte. Plant J 51:281–292

    Article  PubMed  CAS  Google Scholar 

  • Supena EDJ, Winarto B, Riksen T, Dubas E, Van Lammeren A, Offringa R, Boutilier K, Custers J (2008) Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning. J Exp Bot 59:803–814

    Article  PubMed  CAS  Google Scholar 

  • Szűcs A, Jurca M, Jäger K, Barnabás B, Fehér A (2006) Genomic approaches to reveal gene expression changes during fertilization and early seed development in wheat. In: Jäger K, Barnabás B (eds) From gamates to genes. Book of abstracts. XIXth International Congress on Sexual Plant Reproduction. 11–15 July 2006, Budapest, p 65

  • Tahir M, Stasolla C (2006) Shoot apical development during in vitro embryogenesis. Can J Bot 84:1650–1659

    Article  CAS  Google Scholar 

  • Taylor MG, Vasil IK (1995) The ultrastructure of zygotic embryo development in pearl-millet (Pennisetum glaucum, Poaceae). Am J Bot 82:205–219

    Article  Google Scholar 

  • Taylor MG, Vasil IK (1996) The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae). Am J Bot 83:28–44

    Article  Google Scholar 

  • Tereso S, Zoglauer K, Milhinhos A, Miguel C, Oliveira MM (2007) Zygotic and somatic embryo morphogenesis in Pinus pinaster: comparative histological and histochemical study. Tree Physiol 27:661–669

    PubMed  CAS  Google Scholar 

  • Testillano PS, Ramírez C, Domenech J, Coronado MJ, Vergne P, Matthys-Rochon E, Risueño MC (2002) Young microspore-derived maize embryos show two domains with defined features also present in zygotic embryogenesis. Int J Dev Biol 46:1035–1047

    PubMed  CAS  Google Scholar 

  • Thijssen MH, Spoelstra P, Emons AMC (1996) Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays. Physiol Plant 98:539–549

    Article  CAS  Google Scholar 

  • Uchiumi T, Komatsu S, Koshiba T, Okamoto T (2006) Isolation of gametes and central cells from Oryza sativa L. Sex Plant Reprod 19:37–45

    Article  Google Scholar 

  • Uchiumi T, Shinkawa T, Isobe T, Okamoto T (2007a) Identification of the major protein components of rice egg cells. J Plant Res 120:575–579

    Article  PubMed  CAS  Google Scholar 

  • Uchiumi T, Uemura I, Okamoto T (2007b) Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226:581–589

    Article  PubMed  CAS  Google Scholar 

  • van Lammeren AAM (1981) Early events during embryogenesis in Zea mays. Acta Soc Bot Pol 50:289–290

    Google Scholar 

  • Vasilenko A, Mcdaniel JK, Conger BV (2000) Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata. In Vitro Cell Dev Biol Plant 36:51–56

    Article  PubMed  CAS  Google Scholar 

  • Wang YY, Kuang A, Russell SD, Tian HQ (2006) In vitro fertilization as a tool for investigating sexual reproduction of angiosperms. Sex Plant Reprod 19:103–115

    Article  Google Scholar 

  • Yang HP, Kaur N, Kiriakopolos S, McCormick S (2006) EST generation and analyses towards identifying female gametophyte-specific genes in Zea mays L. Planta 224:1004–1014

    Article  PubMed  CAS  Google Scholar 

  • Yeung EC, Stasolla C (2000) Somatic embryogenesis—apical meristems and embryo conversion. Korean J Plant Tissue Cult 27:299–307

    Google Scholar 

  • You R, Jensen WA (1985) Ultrastructural observations of the mature megagametophyte and the fertilization in wheat (Triticum aestivum). Can J Bot 63:163–170

    Article  Google Scholar 

  • Zhang J, Dong WH, Galli A, Potrykus I (1999) Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa). Plant Cell Rep 19:128–132

    Article  CAS  Google Scholar 

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Acknowledgments

The authors wish to thank Emese Békné and Mónika Fehér for their excellent technical assistance. This work was financed by a grant from the National Scientific Research Fund (OTKA K67987) and by a Spanish–Hungarian Cooperation (Project, REF2006HU0004).

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  1. László Szabó

    Present address: Chemical Research Center, Hungarian Academy of Sciences, P.O. Box 17, 1525, Budapest, Hungary

Authors and Affiliations

  1. Plant Cell Biology Department, Agricultural Research Institute, Hungarian Academy of Sciences, P.O. Box 19, 2462, Martonvásár, Hungary

    Ferenc Bakos, László Szabó & Beáta Barnabás

  2. Department of Plant Biochemistry, Cell and Molecular Biology, EEZ (CSIC), Professor Albareda 1, 18008, Granada, Spain

    Adela Olmedilla

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  1. Ferenc Bakos
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  2. László Szabó
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Correspondence to Beáta Barnabás.

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Communicated by Scott Russell.

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Bakos, F., Szabó, L., Olmedilla, A. et al. Histological comparison between wheat embryos developing in vitro from isolated zygotes and those developing in vivo. Sex Plant Reprod 22, 15–25 (2009). https://doi.org/10.1007/s00497-008-0087-7

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