Imaging of native early embryogenic tissue of Scots pine (Pinus sylvestris L.) by ESEM.

Open Life Sci. 2015; 10: 285–290 Nevertheless, complex preparation processes is required to make the sample stable in vacuum conditions, and also electrically conductive. Each step of sample preparation, from chemical fixation, dehydration and drying up to metal coating, can cause various artefact formation and/or destruction of the finer features. High pressure of gas, mostly water vapour in a specimen chamber of the environmental scanning electron microscope (ESEM) together with suitably reduced sample temperature, allows the direct observation of fully hydrated or electrically nonconductive samples in their native state, without the necessity of their surface covering by a conductive layer [7]. It allows to overcome above mentioned SEM disadvantages. In Scots pine (Pinus sylvestris L.) somatic embryogenesis was initiated from immature embryos by Keinonen-Mettälä [8] in 1996. Embryogenic tissue contained first stages of early embryos. Lelu et al. [9] obtained induction and plantlet regeneration from immature seeds. Later, the effect of parent genotype on somatic embryogenesis in P. sylvestris was described. No mother × father interaction was found, indicating that mothers successful at initiation and after 6 months in tissue culture, pollinated by any of the successful fathers, produced somatic lines and mature somatic embryos [10]. Enhancement of somatic embryogenesis from immature zygotic embryos of P. sylvestris and with optimized culture conditions and tight quality control, over 90% of the embryos germinate and develop into vigorously growing emblings [11]. Secondary phenolic compounds (stilbenes) in somatic embryogenesis of P. sylvestris were studied [12]. Somatic embryogenesis is a useful tool to propagate conifers vegetatively. The major limitation in many pine species is the low quality of cotyledonary somatic embryos. The presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development [13]. In the present experiments the new method of environmental scanning electron microscopy (ESEM) was used, which is essentially different from low temperature (LT) ESEM or cryo SEM. The study of the ECMSN with Abstract: Environmental scanning electron microscopy enables the investigation of uncoated pine early embryogenic tissue samples in situ. The samples were examined under low vacuum conditions (air pressure 550 Pa) at a temperature of around -18°C by the AQUASEMII noncommercial environmental scanning electron microscope. The native extracellular matrix surface network was imaged by the environmental scanning electron microscope and in dark field mode of the optical microscope too. The backscattered electron detector disclosed brightness loci in the cells of early embryogenic culture. This work shows images of native pine embryogenic tissues. The continuity of extracellular matrix with structural integrity of plant organism is discussed. Keywords: Somatic embryogenesis, backscattered electron detector, ionization detector, bottle cells, extracellular matrix, structural integrity DOI 10.1515/biol-2015-0028 Received September 23, 2014; accepted March 6, 2015 1 Introduction In several plants cultured in vitro, scanning electron microscope (SEM) analysis revealed that induction of morphogenesis is linked to the appearance of a fibrillar network referred to as the extracellular matrix (ECM) or extracellular matrix surface network (ECMSN). A structural subcellular marker such as ECM was found to be a characteristic feature for embryogenic tissues [1-4]. The ECMSN plays an important morphoregulatory role during somatic embryogenesis and organogenesis, implying an active role in plant morphogenesis [5,6]. Research Article Open Access © 2015 Jiří Hřib et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Jiří Hřib, Božena Vooková*, Vilém Neděla Imaging of native early embryogenic tissue ofScots pine (Pinus sylvestris L.) by ESEM *Corresponding author: Božena Vooková: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, SK-95007 Nitra, Slovak Republic, E-mail: [email protected] Jiří Hřib, Vilém Neděla: Institute of Scientific Instruments, Academy of Sciences of the Czech Republic, CZ-612 64 Brno, Czech Republic 286 J. Hřib et al. higher resolution and larger depth of field than is possible by optical light microscopy can be very important for better description of embryogenic tissue cells in situ. The aim of this study was to present the structure of Pinus sylvestris embryogenic tissue detailed using ESEM studies of native ECMSN. 2 Experimental Procedures 2.1 Induction of Pinus sylvestris embryogenic tissue Immature cones of Pinus sylvestris L. were collected in 2010, June 28th, from trees in Oravský Biely Potok. The zygotic embryos at the time of collection were at the precotyledonary stage of development. Megagametophytes containing embryos were cultured on DCR medium [14] containing 0.5 mg L-1 BAP, 2 mg L-1 2,4dichlorophenoxyacetic acid (2,4-D), 200 mg L-1 myoinositol, 500 mg L-1 casein hydrolysate, 50 mg L-1 glutamine, 20 g L−1 sucrose, solidified with 3 g L−1 Phytagel. The pH was adjusted to 5.8 before autoclaving. The medium was poured into Petri dishes (6 cm in diameter). The cultures were grown in darkness at 25 ± 1°C. Induction of Pinus sylvestris embryogenic tissue was observed after five week cultivation of immature zygotic embryos on initiation medium. This tissue contained mucilaginous cell mass proliferated from the micropylar end of megagametophyte. Proliferation occurred on the same initiation medium in darkness. The tissue was transferred to a fresh medium at 2 - 3 week intervals. For the observation in ESEM the 30th subculture of embryogenic tissue was used (Fig. 1). 2.2 Observation in ESEM Native samples (free of chemical fixation and drying) of embryogenic tissue in proliferation stage cultured on fresh medium during 10 days, were placed on a cooled specimen holder (Peltier stage) and their temperature was gradually decreased and then maintained between -18 and -22°C. Due to the relatively low heat conductivity of the conifer samples (dimensions of 2–3 mm2 and thickness of 2 mm), the real temperature of the sample surface can be higher. At the beginning of the pumping the pressure was equal to the atmospheric pressure in all parts of the microscope. The pumping process started approximately one minute after the decrease of the sample holder temperature. Imaging was started after ten minutes when the sample was inserted. Every one of selected parts were observed approximately 8-10 minutes. Whole experiment was finished after 30 minutes including manipulation and imaging. Described method was tested on 36 sites of early embryogenic tissue. Paper shows results of two observed series each contains four samples. The conifer samples were examined under low vacuum conditions (air pressure 550 Pa) by the AQUASEM II non-commercial ESEM. It was designed in the Institute of Scientific Instruments of the Academy of Sciences of the Czech Republic for research on detection systems and ESEM techniques [15]. The single crystal YAG:Ce3+, used for detection of backscattered electrons (BSEs) in high and low vacuum conditions, has a hole in the center so that it simultaneously acts as a pressure-limiting aperture of the AQUASEM II [14]. Generally, the system of pressure limiting apertures in the ESEM enables maintaining low pressure (from 10-3 to 10-6 Pa) of a gas in the vicinity of the electron source and simultaneously, the use of relatively high pressure of the gas (from 1 to approx. 2000 Pa) in the specimen chamber of the microscope. In low vacuum and environmental modes the secondary electron (SE) signal can be detected by an ionization detector using an electrode with the inner diameter of 4 mm deposited on the input surface of the YAG single crystal. With a positive bias of 280 V with respect to the sample, it acts in a similar fashion to the environmental secondary electrons detector. The combination of the BSE-YAG detector of highenergy BSEs emitted from deep layers of the sample and thus yielding information on the material contrast (electron emission is strongly dependent on the atomic number of the sample) with a special ionization detector [16] recording signals composed of predominantly lowFig. 1: Early embryogenic tissue of Scots pine (Pinus sylvestris L.) in the dark field (Olympus Lext OLS 3100) (the presence of the mucilaginous matrix is indicated by arrows, the present of head is indicated by *). Imaging of native early embryogenic tissue of Scots pine (Pinus sylvestris L.) by ESEM 287 energy SE emitted from the surface layers of the sample and giving topographic contrast enables us to detect information on the surface structure of the studied samples, and moreover on their material composition. All experiments were carried out under constant operating conditions (beam accelerating voltage 20 kV, probe current 70 pA, sample distance 2.5 mm between the bottom surface of the YAG single crystal and the surface of the sample, positive bias of the detection electrode system 280 V) and in the gas environment with relative humidity equal to 40%. 3 Results and Discussion Study of P. sylvestris embryogenic tissue (embryonal suspensor mass) by the methodology of natural low temperature ESEM showed that this tissue contained long suspensor cell aggregates (Fig. 2A,B), aggregates of bottle shape cells (Fig.3 A-F) and early somatic embryos composed from embryonal heads (Fig. 4A-F) with suspensor cells. It was observed that suspensor cells in early somatic embryos of Scots pine play important roles in embryo development [13]. The surfaces of the bottle shape cells as well as the embryonal heads were covered with mucilaginous matrix – ECM (ECMSN). It seems that consistency of this matrix is different from the networklike or fibrillar structure observed in other species [4,17,18]. Consistent with Lai et al. [4] we suppose that the ECM layer forms bridges connecting the neighboring cells together and may be the early structural marker for embryogenic tissues. Bottle shape cells were described by Šamaj et al. [18] as well developing somatic embryos. It is interesting that bottle cells in animals contribute significantly to gastrulation. The role of bottle-shaped cells in Scots pine somatic embryogenesis in not known. The most readily observable result of the interaction of cells with the ECM is cell adhesion. The adhesion of plant cells to one another is fundamental to the formation and maintenance of plant structure [19]. Our experiments indicate significant adhesion of early embryogenic tissue of Pinus sylvestris. Hoenemann et al. [20] described that in the nonembryogenic cell line of Cyclamen persicum the much looser cell adhesion might be caused by reduced pectin content, which might be an important factor for the loss of embryogeneity due to reduced cell adhesion. It is probable that in the conifers, cell adhesion is important in the process of Pinus somatic embryogenesis. The BSE detector allowing for images with contrast relating to the atomic weight of the elements in the sample was used in this study. The signal of BSEs has been widely used for the investigation of specimen surface in scanning electron microscopy for many years [21,22]. A new type YAG – BSE detector for high resolution SEM imaging of large biological samples was presented [23]. A variable-pressure scanning electron microscope (VPSEM) equipped with a high-sensitive BSE detector of the YAG type was applied to studies of fixed biological tissue samples [24]. Our micrographs (Fig. 2B; 3B, D, F; 4B, D, F) are from ESEM on non-fixed samples with a BSE-YAG detector. Other subsequent experiments display more accurate determination of elements in the cells of pine Fig. 2: ESEM observations of early embryogenic tissue (Pinus sylvestris L.). (Ausing ionization detector, Busing the BSE YAG detector (accelerating voltage 20 kV, probe current 70 pA, ionization detector 270 V, pressure of air in the specimen chamber 550 Pa), the presence of the ECMSN is indicated by full arrows, S-long suspensor cells). 288 J. Hřib et al. early embryogenic tissue which appeared as brightness loci (Fig. 3 D, F). Preliminary our experiments suggest the displaying of calcium and potassium distributions in the cells. The interactions between cells play a fundamental role in the growth and development of multicellular organisms. Structural integrity of plants was described by Šebánek et al. [25]. It may also be mentioned that the Fig. 3: ESEM observations of bottle shape cells in Scots pine (Pinus sylvestris L.) somatic embryogenesis. (A,C,E - using ionization detector, B,D,F - using the BSE YAG detector; the presence of the ECMSN is indicated by full arrows, the presence of brightness loci by dashed arrows). Imaging of native early embryogenic tissue of Scots pine (Pinus sylvestris L.) by ESEM 289 extracellular matrix issue pertains to Dostal´s [26] topic of structural integrity of plant organism. This paper introduces the native structure of pine embryogenic tissues investigated using electron microscope. For the future, the use of the methodology with low temperature presented in this work is offered for the study of organisms for example algae, lichen and following directly from Arctic or Antarctic nature and with it, to apply this also generally in polar biology. Fig. 4: ESEM images of pine embryogenic head. (A,C,E - using ionization detector, B, D, F - using the BSE YAG detector; the presence of the ECMSN is indicated by full arrows, the presence of own heads by *). 290 J. Hřib et al. Acknowledgements: This work was supported by the Grant Agency of the Czech Republic, grant No. GA1422777S and MEYS CR (LO1212) together with EC (ALISI No. CZ.1.05/2.1.00/01.0017) and by AS CR (RVO:68081731) and Slovak Grant Agency VEGA, Project No. 2/0136/14. This work was also co-funded by the European Community under project No. 26220220180, Building Research Centre “AgroBioTech”. Conflict of interest: Authors declare nothing to disclose. References [1] Dubois T., Dubois J., Guedira M., Diop A., Vasseur J., SEM characterization of an extracellular matrix around somatic proembryos in roots of Cichorium, Ann. Bot., 1992, 70, 119–124 [2] Jásik J., Salajová T., Salaj J., Developmental anatomy and ultrastructure of early somatic embryos in European black pine (Pinus nigra Arn.), Protoplasma, 1995, 185, 205–211 [3] Šamaj J., Bobák M., Blehová A., Krištin J., Auxtová - Šamajová O. , Developmental SEM observations of an extracellular matrix in embryogenic calli of Drosera rotundifolia and Zea mays, Protoplasma, 1995, 186, 45–49 [4] Lai K.S., Yusoff K., Maziah M., Extracellular matrix as the early structural marker for Centella asiatica embryogenic tissues, Biol. Plantarum, 2011, 55, 549–553 [5] Šamaj J., Baluška F., Bobák M., Volkmann D., Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4, Plant Cell Rep., 1999, 18, 369–374 [6] Šamaj J., Bobák M., Blehová A., Preťová A., Importance of cytoskeleton and cell wall in somatic embryogenesis. In: A. Mujib, J. Šamaj (Eds.), Plant Cell Monographs, Vol. 2 - Somatic embryogenesis, 1st ed., Berlin, Heidelberg, Springer-Verlag, 2006, 35-50 [7] Popielarska-Konieczna M., Bohdanowicz J., Starnawska E., Extracellular matrix of plant callus tissue visualized by ESEM and SEM, Protoplasma, 2010, 247, 121-125 [8] Keinonen-Mettälä K., Jalonen P., Eurola P., von Arnold S., von Weissenberg K., Somatic embryogenesis of Pinus sylvestris, Scand. J.For. Research, 1996, 11, 242–250 [9] Lelu M.A., Bastien C., Drugeault A., Gouez M.L., Klimaszewska K., Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on the medium with and without plant growth regulators, Physiol. Plantarum, 1999, 105, 719–728 [10] Niskanen A.M., Lu J., Seitz S., Keinonen K., von Weissenberg K., Pappinen A., Effect of parent genotype on somatic embryogenesis of Scots pine (Pinus sylvestris), Tree Physiol., 2004, 24, 1259-1265 [11] Aronen T., Pehkonen T., Ryynänen L., Enhancement of somatic embryogenesis from immature zygotic embryos of Pinus sylvestris, Scand. J. Forest Research, 2009, 24, 372-383 [12] Lu J., Konstianen K., Jaakola L., Heiska S., Harju A., JulkunenTiitto R.,Venäläinen M., Aronen T. Secondary phenolic compounds in somatic embryogenesis of Pinus sylvestris L. – a preliminary study. In: Y.S. Park, J.M. Bonga (Eds.), Integrating vegetative propagation, biotechnologies and genetic improvement for tree production and sustainable forest management, Proceedings of the IUFRO Working Party 2.09.02 conference, June 25-28, 2012 Brno, Czech Republic, Session 4, p. 1 [13] Abrahamsson M., Valladares S., Larsson E., Clapham D., von Arnold S., Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death, Plant Cell Tiss Organ Cult., 2012, 109, 391-400 [14] Gupta P.K., Durzan D.J., Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana), Plant Cell Rep., 1985, 4, 177-179 [15] Neděla V., Controlled dehydration of a biological sample using an alternative form of environmental SEM, J.Microsc., 2010, 237, 7-11 [16] Neděla V., Konvalina I.; Lencová B.; Zlámal J., Comparison of calculated, simulated and measured signal amplification in a variable pressure SEM, Nucl. Instr. & Methods. in Physics Research, Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 2011, 645, 79-83 [17] Pilarska M., Knox J.P., Konieczny R., Arabinogalactan-protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in Trifolium nigrescens Viv., Plant Cell Tiss.Organ Cult. 2013, 115, 35-44 [18] Šamaj J., Salaj T., Matúšová R., Salaj J., Takáč, T., Šamajová O.,Volkmann D., Arabinogalactan-protein epitope Gal4 is differentially regulated and localized in cell lines of hybrid fir (Abies alba x Abies cephalonica) with different embryogenic and regeneration potential, Plant Cell Rep., 2008, 27, 221-229 [19] Waldron K.W., Brett C.T., The role of polymer cross-linking in intercellular adhesion, In: J.A. Roberts, Z. Gonzalez-Carranza (Eds.), Plant Cell Separation and Adhesion, Annual Plant Reviews, Volume 25, Blackwell Publishing Ltd., 2007, 183-204 [20] Hoenemann C., Richardt S., Krüger K., Zimmer A.D.., Hohe A., Rensing S.A. Large impact of the apoplast on somatic embryogenesis in Cyclamen persicum offers possibilities for improved developmental control in vitro, BMC Biology, 2000, 10,77 doi: 10.1186/1471-2229-10-77 http:// www.biomedcentral.com/14712229/10/77 [21] Wells O.C., Backscattered electron image (BSI) in the scanning electron microscope (SEM), Scanning Electron Microsc., 1977, 1, 747-771 [22] Ball M.D., Mc Cartney D.G., The measurement of atomic number and composition in an SEM using backscattered detectors, J.Microsc., 1981, 124, 57-68 [23] Walther P., Autrata R., Chen Y., Pawley J.B., Backscattered electron imaging for high resolution surface scanning electron microscopy with a new type YAG-detector, Scanning Microsc., 1991, 5, 301-310 [24] Ushiki T., Hashizume H., Itoh S., Kuboki K., Saito S., Tanaka K., Low-voltage backscattered electron imaging of non-coated biological samples in a low vacuum environment using a variable-pressure scanning electron microscope with a YAG-detector, J. Electron Microsc. (Tokyo), 1998, 47, 351-354 [25] Šebánek J., Sladký Z., Procházka S., Luxová M., Erdelská O., Experimental Morphogenesis and Integration of Plants, 1st ed., Academia Praha, published in co-edition with ELSEVIER, 1991 [26] Dostál R., On Integration in Plants, 1st ed., Harvard University Press, 1967