Abstract
Main conclusion
Microscopic techniques remain an integral tool which has allowed for the better understanding and manipulation of in vitro plant culture systems. The recent advancements will inevitably help to unlock the long-standing mysteries of fundamental biological mechanisms of plant cells.
Beyond the classical applications in micropropagation aimed at the conservation of endangered and elite commercial genotypes, plant cell, tissue and organ cultures have become a platform for elucidating a myriad of fundamental physiological and developmental processes. In conjunction with microscopic techniques, in vitro culture technology has been at the centre of important breakthroughs in plant growth and development. Applications of microscopy and plant tissue culture have included elucidation of growth and development processes, detection of in vitro-induced physiological disorders as well as subcellular localization using fluorescent protein probes. Light and electron microscopy have been widely used in confirming the bipolarity of somatic embryos during somatic embryogenesis. The technique highlights basic anatomical, structural and histological evidence for in vitro-induced physiological disorders during plant growth and development. In this review, we discuss some significant biological insights in plant growth and development, breakthroughs and limitations of various microscopic applications and the exciting possibilities offered by emergent in vivo live imaging and fluorescent protein engineering technologies.
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Introduction
The concept and discovery of microscopy date back approximately four centuries. Basically, microscopy involves the use of microscopes to enlarge samples or objects which originally are beyond the resolution of the human eye (Shur and Price 2012; Thomasson and Macnaughtan 2013). As an indication of the great importance and value of microscopy, it remains a popular and vital tool with a wide range of applications in basic and applied sciences (Tranfield and Walker 2013; Zumbusch et al. 2013; El-Bakry and Sheehan 2014; Whited and Park 2014) as well as in the medical and material engineering fields (Torrealba and Carrasco 2004; Shur and Price 2012; De Boer et al. 2013; Juszczyk et al. 2013). These aforementioned publications also provide excellent reviews highlighting the trends and current updates related to the specifics and significance of microscopy in these various fields. In addition, details on the theory and practice of specific microscopy-based technologies and specimen preparation protocols are well documented (Chalfie and Kain 2005; Kuo 2007; Chandler and Roberson 2009; Murphy and Davidson 2013).
In plant sciences, microscopy is used in an attempt to resolve and understand various aspects of growth and developmental processes including structural and functional properties. It also provides insights on interactions of cells and subcellular components in plants (Chandler and Roberson 2009; Domozych 2012). Plant science as a field encompasses diverse aspects of plant growth, development and ecology amongst others. With one of the main focus geared at efficient propagation and general plant improvement in terms of quality and quantity, plant biotechnology remains one of the fundamental fields in plant sciences (Vasil 2008). To a certain extent, plant biotechnology was established based on the principles of cellular totipotency and genetic transformation. Inevitably the use of basic in vitro plant culture techniques is essential and vital for the success of several plant biotechnology endeavours. Particularly from a conservation perspective, the value and benefit of in vitro tissue culture systems (micropropagation) are well documented (George 1993; Ramachandra Rao and Ravishankar 2002; Pence 2010). Besides the potential in ensuring food security via mass propagation of different staple crops and fruits (Mondal et al. 2004; Dobránszki and Teixeira da Silva 2010), many plant species with ornamental, horticulture and medicinal values are easily regenerated (Rout et al. 2000, 2006; Teixeira da Silva 2003; Moyo et al. 2011), see Fig. 1.
Recent technological advances are expanding the capabilities of microscopy which are being used to understand and explain commonly observed morphological appearances of in vitro regenerants. Thus, coupled with complementary biochemical, histological and molecular approaches, the increasing diverse microscopy-based technologies can expedite the better understanding of in vitro plant culture systems. Furthermore, physiological disorders in regenerants are often better elucidated with the use of microscopic systems (Chakrabarty et al. 2006; Jausoro et al. 2010a; Bairu et al. 2011). Notwithstanding these aforementioned applications and benefits, a comprehensive review detailing the contribution of microscopy to the understanding of in vitro plant culture systems is still lacking. Thus, this review covers a brief overview on the basic microscopic principles and technological advances in the field as well as summarizing the present applications and gains from the use of microscopy in in vitro plant culture systems. In addition to identifying the current knowledge gap, a critical appraisal of microscopy application in plant cell, tissue and organ culture systems was discussed. Even though the current review is not fully exhaustive of all the available literature, as much as possible, we provide representative and specific references to ascertain the overall objectives of the subject matter.
Recent advances and general overview on microscopic techniques
In recent times, novel and giant technological strides in the form of introduction of laser-based, vibrational, electron and X-ray systems coupled with the rapid evolution of digital image capture and analysis technologies have revolutionized the capabilities and applications of microscopy (Torrealba and Carrasco 2004; Roberts et al. 2007; Domozych 2012; Picas et al. 2012). As postulated by Domozych (2012), these developments have allowed for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. Based on the evidence of its increasing application (Jahn et al. 2012; Picas et al. 2012; Thomasson and Macnaughtan 2013; Zumbusch et al. 2013; El-Bakry and Sheehan 2014; Whited and Park 2014), there is no doubt that microscopy is more valuable than ever before and will remain relevant in all areas of plant science research. In the near future, modern microscopy will ultimately achieve the goal of resolving the three-dimensional (3-D) structural and functional features of cellular life (four-dimensional imaging or 4-DI) (Domozych 2012).
Despite the potential and advances associated with microscopic techniques for biological research, some inherent limitations still exist. The two major ‘reality checks’ are that (1) light microscopy and confocal laser scanning microscopy (CLSM) used to image dynamic events in live cells are inherently limited in resolution and (2) electron microscopy which possesses better resolution cannot be used to view live cells (Domozych 2012). Although scanning probe microscopy exists as a different technique, optical (light) and electron microscopy are the most commonly used in in vitro culture systems (Tables 1, 2, 3). The conventional light microscope techniques are bright-field, polarized, and fluorescence light microscopy while electron microscopy includes the scanning electron microscopy and transmission electron microscopy (TEM) (Chandler and Roberson 2009; Domozych 2012). In addition to other basic differences such as sample preparation, electron microscopy has a much higher resolution of ≈0.1–5 nm than light microscopy with a resolution of 0.2 mm (El-Bakry and Sheehan 2014). Thus, the task to be performed and specific objective generally influence the choice of microscopy technique at any given time.
Practical application of microscopic techniques in in vitro plant culture systems
As a well-established system for rapid proliferation of clonal plantlets (Fig. 1) for the floricultural and ornamental industries, micropropagation allows for year-round and continuous culture (Caponetti et al. 2005; Pence 2010; Ruffoni and Savona 2013). For plant species with medicinal value, their mass propagation is often aimed at ensuring their conservation (Canter et al. 2005). Furthermore, researchers have in recent times unravelled the potential of plant tissue culture as a tool to elucidate metabolic pathways and to enhance the production of therapeutic phytochemicals (Ramachandra Rao and Ravishankar 2002; Verpoorte and Memelink 2002; Karuppusamy 2009). In vitro cell culture systems are also valuable avenues for transformation and transgenic studies. The following subsections highlight the basic and practical application of microscopy in different in vitro systems.
Elucidation of growth and development patterns
Even though the frequency of application may differ, the four basic methods for micropropagation are axillary shoot proliferation, node culture, de novo formation of adventitious shoots through organogenesis and somatic embryogenesis (Kane 2005). Details of these procedures are outside the scope of this review but have been well documented (Zimmerman 1993; Kane 2005; Rout et al. 2006). There is no doubt that much of the available evidence and theories of in vitro developmental processes were achieved via histological approaches using different microscopic techniques (Trigiano et al. 2005). Although tremendous advances have been recorded in recent times (Motte et al. 2014), more stringent studies are required to fully understand the overall intricate events involved in in vitro plant growth and development. With the ability to generate microscopic structures and characteristics of cells through initiation, assemblage and arrangement phases, researchers have gained in-depth knowledge which allows for manipulation of plant growth using in vitro culture techniques. Both light and electron microscopic techniques have become integral components in studying plant species (Table 1). Plant growth and development processes are characteristically dynamic whereas histological techniques only present a narrow momentary glimpse of the process (Trigiano et al. 2005). Notwithstanding, by piecing together a series of the static microscopic observations, plant biologists are able to elucidate the underlying anatomical features involved in plant development.
Somatic embryogenesis
The induction of somatic embryos under in vitro culture conditions in ontogenetic steps is similar to those observed in zygotic embryogenesis and has long fascinated plant biologists (Zimmerman 1993). Somatic embryogenesis (SE) provides a model system for studying the genetic basis of early differentiation events and cellular totipotency of somatic cells (Zimmerman 1993; Fehér et al. 2003; Kurczyńska et al. 2007). Furthermore, SE has become a widely used technique in genetic transformation and mass propagation of elite genotypes (Table 1). Light and electron microscopic studies have reported on the cellular origin of somatic embryos during primary (Blazquez et al. 2009; Capelo et al. 2010; Lin et al. 2011; Parra-Vega et al. 2013) and repetitive/secondary embryogenesis (Dai et al. 2011; Pavlović et al. 2013; Raju et al. 2013). Upon exposure to SE induction medium, the initial events signifying cytological changes in the formation of somatic embryos were evident in 4–6 days (Canhoto et al. 1996; Kurczyńska et al. 2007). Induction of somatic embryos was characterized by formation of embryonic-like centres from single or multi-cells of proto- and subprotodermal origin (Kurczyńska et al. 2007), accumulation of starch grains and differentiation of mitochondria (Canhoto et al. 1996). The unicellular origin of direct somatic embryos has been observed to be the main morphogenic pathway (Rugkhla and Jones 1998; Kurczyńska et al. 2007). Thus, as early as 6 days into the culture period, SE induction can be confirmed, especially for somatic embryo transformation studies. Furthermore, microscopic applications coupled with molecular techniques provides an invaluable OMICS/morphology interface for exploring plant growth and development. During the early stages of embryogenesis, LEAFY COTYLEDON (LEC) genes play a key role in somatic embryo development (Stone et al. 2001). In Arabidopsis thaliana, strong promoter activity of LEC was detected at the globular somatic embryo stage after a 12-day culture period (Kurczyńska et al. 2007). In addition, microscopic techniques have been highly valuable in characterizing the non-bipolarity of protocorm-like bodies (Mayer et al. 2010; Huang and Chung 2011), globular embryo-like structures (Woo and Wetzstein 2008; Sharifi et al. 2010) and nodular meristemoids (Moyo et al. 2009; Rosa and Dornelas 2012), which would have been otherwise mistaken for somatic embryos (Fig. 2). Thus, microscopy techniques have been invaluable in confirming and ascertaining the bipolar identity of somatic embryos in SE or lack thereof in embryo-like structures during organogenesis.
Organogenesis
Organogenesis refers to de novo organ formation involving the processes of dedifferentiation and redifferentiation of plant cells. It is widely proven that the ratio of auxin to cytokinin in plant tissues has the ability to shift the cell physiological state. In particular, the distribution and unique movement of auxins in a polar direction from cell to cell is thought to have a major influence on the organogenic fate of plant tissues (Muday and DeLong 2001; Del Bianco et al. 2013; Motte et al. 2014). However, the underlying mechanisms involved in this process remain to be fully elucidated (Motte et al. 2014). Innovative approaches using reporter genes fused to specific promoters, such as peptidyl-prolyl cis/trans isomerase (PIN1) and microscopy techniques have attempted to decipher the physiological and molecular mechanisms controlling the process of organogenesis (Vieten et al. 2007). When used in conjunction with plant tissue culture model systems, the use of visual markers such as β-glucuronidase (GUS), luciferase (LUC), β-galactosidase (LacZ) and green fluorescent protein (GFP) could be useful in exploring the molecular mechanisms controlling plant growth and development. In plant transformation studies, GFP allows for non-destructive direct observation of gene expression events and the successful recovery of transgenic plants (Hraška et al. 2006). Therefore, microscopy coupled with plant tissue culture offers a platform for molecular/morphology assessments in plant growth and development studies.
Furthermore, regardless of the plant cell, tissue and organ culture technique, establishing the specific origin of structures such as adventitious shoots, roots and somatic embryos remains one of the unsolved mysteries in plant biology (Trigiano et al. 2005). Numerous microscopy studies have provided significant insights into the basic structural features of in vitro plant growth (Table 1). For example, using light microscopy, the regeneration rate of hypocotyl subsections (C1–C4) in Watsonia lepida showed that cell division was highest in C2 while in vitro regeneration was significantly lower than in subsection C1 (Ascough et al. 2009). The authors reported that subsection C1 contained the apical meristem which possibly had meristematic cells that are developmentally plastic and responsive to external cues. Using optical microscopy da Cruz et al. (2014) showed that cell proliferation within the pericycle led to adventitious bud formation in Bixa Orellana root explants. Rocha et al. (2012) characterized the anatomical events and ultrastructural aspects involved in Passiflora edulis direct and indirect in vitro organogenesis. The study showed that irrespective of the organogenic process, P. edulis meristemoids had similar ultrastructural characteristics. These and other similar findings provide increased knowledge and critical insights that allow a better understanding of in vitro organogenic processes.
Other cellular developmental patterns
Furthermore, light and electron microscopy have contributed immensely in evaluating the effects of various physiological factors on cellular developmental processes (Table 1). Nakagawa et al. (2011) demonstrated that the distribution of starch grains during SE was induced by exogenous application of polyamines. In addition, spermine-treated Triticum aestivum plants exhibited smaller chloroplasts compared to putrescine and spermidine-treated ones, suggesting that the response was dependent on the type of polyamine (Redha and Suleman 2011). In particular, TEM provides ultrastructural details of cellular developments when plants are exposed to different physiological stimuli. Using TEM, López-Villalobos et al. (2011) showed that lauric acid induced the production of large oil bodies and a high number of organelles in Cocos nucifera zygotic embryos. The scope for elucidating the ultrastructural developmental patterns arising from physiological stimuli remains limitless.
Detection of in vitro-induced physiological and anatomical disorders
Despite the benefits of micropropagation, the process is often besieged by a number of in vitro-induced challenges which may be anatomical, physiological and biochemical in nature (Kaeppler et al. 2000; Hazarika 2006; Bairu et al. 2011; Neelakandan and Wang 2012; Ruffoni and Savona 2013). Several studies have demonstrated the effects of the controlled, largely artificial environment in plant tissue culture systems on the anatomy of in vitro plants (Figs. 3, 4). Researchers have continuously reviewed the subject matter (Bairu and Kane 2011) and suggested means of tackling the recurrent problems such as shoot-tip necrosis (Bairu et al. 2009), hyperhydricity (Ziv 1991; Rojas-Martínez et al. 2010), fasciation (Iliev and Kitin 2011), epigenetic (Kaeppler et al. 2000; Smulders and de Klerk 2011) and somaclonal variations (Larkin and Scowcroft 1981; Bairu et al. 2011). Most of these physiological disorders are not only limited to the period of in vitro growth but become more apparent upon acclimatization of the regenerants (Kozai 1991; Hazarika 2006; Pospíšilová et al. 2007). As a result, the success of plant tissue culture especially on a large scale depends on how these challenges can be alleviated or possibly eradicated (Kozai et al. 1997; Hazarika 2006; Bairu and Kane 2011). A better understanding of these multifaceted problems begins with the availability of appropriate identification tools. In view of the substantial evidence (Table 2), there is no doubt that the application of microscopy remains critical in understanding these challenges. In addition, other approaches such as biochemical and molecular tools provide complementary evidence for overall elucidation of the problems. The importance of microscopy is possibly attributed to the fact that the majority of physiological disorders are often manifested in the anatomy of the tissue-cultured regenerants.
Both light and electron microscopic techniques have demonstrated vital significance in the attempt to elucidate the anatomical and histological basis for in vitro-induced physiological disorders in several plant species (Table 2). Amongst these in vitro-induced challenges, detection of hyperhydricity has received considerable success with the use of different microscopic techniques (Table 2). Considering that hyperhydricity affects several organelles in the cells of regenerated plants, it becomes necessary to examine the structure for detection of possible aberrations. With the use of light microscope, parameters such as surface wax topology and leaf imprints are recorded while the epidermal and stomatal cell count can be easily achieved (Correll and Weathers 2001). Variations in these aforementioned parameters afford for direct evidence on the possible underlying metabolic processes which are responsible for the incidence of hyperhydricity in regenerated plants. Evidence from scanning electron microscopy revealed that thickening of the stem and retardation of elongation are the first changes observed in hyperhydric carnation plantlets (Werker and Leshem 1987). Examining the ultrastructural differences between the hyperhydric and normal leaves of carnation plantlets, Olmos and Hellin (1998) observed large vacuolated mesophyll cells (showing hypertrophy of cells and large intercellular spaces), lack of cuticular wax and the presence of abundant plastoglobuli on chloroplasts in hyperhydric leaves. The authors also noted differences in the morphology of guard cells with X-ray microanalysis revealing high levels of K+ on abnormal plants. Furthermore, stomatal density was significantly greater in normal leaves while the crystalline structure of the epicuticular wax was absent in hyperhydric leaves. An irregular assortment of organelles and unorganized spongy mesophyll were also observed in hyperhydric leaves.
With studies involving the use of TEM for hyperhydricity, critical examination of the ultrastructure of plant cells and tissues remains the main objective. In such instances, organelles such as the chloroplast and mitochondria are often the main focus of researchers. In Allium sativum, hyperhydric cells had swollen mitochondria and slender chloroplasts (Wu et al. 2009). The authors also observed that the vacuole displaced the organelle to the cell wall edge and the intergranal thylakoids appeared compressed. While the structure of mitochondria and peroxisomes did not change in hyperhydric Capsicum annuum plants, the number of peroxisomes was more than in normal plants (Fontes et al. 1999). Furthermore, the chloroplasts in the hyperhydric plants exhibited thylakoid disorganization, low grana number as well as presence of large starch grains and a low accumulation or absence of plastoglobules.
Subcellular localization and characterization
The benchmark discovery of the wild-type green fluorescent protein (GFP) from the jellyfish Aequorea victoria (Shimomura et al. 1962), cloning of the GFP gene (Prasher et al. 1992), and its modification into a functional fluorescent protein (Chalfie et al. 1994) have revolutionized the study of plant cell biology. However, variable outcomes have been reported with the expression of the wild-type gfp gene in different plants. The expression of gfp in Arabidopsis and other plant species was shown to be curtailed by aberrant mRNA splicing in which an 84 nucleotide sequence, recognized as a cryptic intron, codes for a defective protein (Haseloff and Siemering 2005). A modified gfp gene without the cryptic intron sequence exhibited improved in vivo expression in a wide range of plant species (Reichel et al. 1996). Further improvements in sensitivity of the marker protein have been achieved through modifications of the GFP mutant cDNA leading to single-amino acid exchanges in the chromophore region. Green fluorescent protein and its derivative fluorophores have emerged as important reporter proteins for monitoring gene expression (Tang et al. 2005; Rosa et al. 2013; Yang et al. 2013), subcellular protein localization (Huai et al. 2009; Lai et al. 2013; Liu et al. 2013), organelle dynamics (Hashimoto et al. 2011; Tewari et al. 2013; Xu et al. 2013) and cell transformation (Holme et al. 2006), both in vivo and real time, as well as in fixed samples (Davidson and Campbell 2009). Furthermore, a combination of plant cell, tissue and organ culture techniques and fluorescent protein tags has provided a powerful tool for unravelling fundamental insights into mechanisms involved in plant morphogenesis. In vitro plant culture provides an ideal environment that can be precisely controlled and modified to achieve specific experimental conditions. Thus, the application of fluorescent probes in plant tissue culture systems has elucidated developmental and molecular mechanisms involved in plant morphogenic processes (Table 3). The most commonly used fluorescent probe application is probably protein tagging for monitoring dynamic cellular events and subcellular protein localization using confocal laser microscopy (Sirerol-Piquer et al. 2012). In addition, dynamic expression patterns of fluorescent probes have revealed interesting spatial and temporal changes in morphogenic events involving plant cell, tissue and organ culture processes such as SE (Ramakrishna et al. 2012; Bouchabké-Coussa et al. 2013) and embryonic cell suspension cultures (Cole et al. 2013).
Beyond the resolution limits of light microscopy, GFP immunogold TEM provides more detailed information on subcellular localization of proteins. The high-resolution property of TEM allows for the detection of immunogold labelled GFP-tagged proteins in the cytoplasm, organelles and plasma membrane (Boevink et al. 1998; Nebenführ et al. 1999; Follet-Gueye et al. 2003). Using this immunocytochemical technique, Potocka et al. (2012) demonstrated spatial and temporal changes in the distribution of lipid transfer protein epitopes during SE. However, the technique has only been sparsely applied in studying morphogenesis in plant cell, tissue and organ cultures (Table 3). Notwithstanding benefits derived from the high resolving power of immunoelectron microscopy, the technique has inherent drawbacks such as preservation of GFP antigenicity and antibody specificity, arising from denaturization and bleaching of GFP during polymerization; decreased immunogold staining with tissue depth (Sirerol-Piquer et al. 2012) as well as safety concerns associated with the use of uranyl acetate in specimen preparation (Carpentier et al. 2012). In attempts to find alternatives for uranyl acetate, polyphenolic compounds such as tannic acid (Kajikawa et al. 1975) and oolong tea extracts (Sato et al. 2008; Carpentier et al. 2012) have been evaluated for staining ultrathin sections. Other recent protocols using microwave-assisted processing resulted in good preservation of cell antigenicity and high-quality cell ultrastructure for immunocytochemical studies (Carpentier et al. 2012). Polyphenol-containing extracts, for example oolong tea extracts exhibited good counterstaining properties for both ultrathin sections and in block staining, making them possible alternatives for the hazardous heavy metal stains such as uranyl acetate and lead citrate. Notwithstanding, specimen fixation with glutaraldehyde and osmium tetroxide (OsO4), and double electron staining with uranyl acetate and lead salts provide excellent contrast enhancement, hence it has remained standard procedure in most microscopy laboratories (Sato et al. 2008). Thus, until the discovery of suitable alternatives, common stains such as uranyl acetate, uranyl formate, methylamine tungstate and methylamine vanadate will continue to be used but with emphasis on observance of safety regulations.
The search for fluorophores with low phototoxicity and decreased autofluorescence has advanced the boundaries of fluorescent protein engineering. Together with the development of high-resolution imaging techniques, a range of fluorescent protein probes with diverse spectral qualities spanning the orange, red and far-red regions of the electromagnetic spectrum have been developed (Davidson and Campbell 2009). Some studies (Smith-Espinoza et al. 2007; Wu et al. 2011; Sun et al. 2013) used enhanced GFP (eGFP), a variant of the GFP mutant in which exchange of amino acid phenylalanine 64 to leucine (F64L) and serine 65 to threonine (S65T) drastically increased brightness intensity and photostability (Reichel et al. 1996; Zacharias and Tsien 2005). Modifications of GFP have resulted in some variants with better fluorescence characteristics, for example the maturation of eGFP is four times faster than that of the wild type (Ckurshumova et al. 2011).
Conclusions and future perspectives
The discovery and advancement of microscopic technologies have provided plant biologists with a wide array of invaluable techniques to explore cellular structures and dynamics, thereby expanding our knowledge of plant growth and development. When used in conjunction with plant cell, tissue and organ culture methods, microscopic applications have provided critical insights into the dynamics of plant growth and development. In particular, the live imaging capabilities afforded by confocal microscopy and fluorescent protein probes (GFP and its derivatives) have further advanced the boundaries in plant morphogenesis research and expanded the possibilities of what can be achieved in the future with improved resolving power of light microscopy. Development of photostable fluorophores, especially in the red and far-red spectral regions will provide more biological insights through dynamic in vivo live imaging of cellular components. Thus, notwithstanding the limited resolving power of light microscopy, the ‘illuminated plant cell’ (Mathur 2007) continues to contribute invaluable information on subcellular protein localization, gene expression and transport of molecules, thereby enhancing our understanding of the fundamental mechanisms involved in plant developmental process. Furthermore, immunoelectron microscopy and immunogold labelling have circumvented the drawbacks imposed by the limited resolving power of light microscopy. New advancements and novel innovations in specimen preparation techniques’ using high-phenol content plant extracts such as OTE (in place of uranyl acetate) and microwave-assisted processing are likely to expand the utilization of this method. Despite having high resolving power, immunogold labelling using TEM is still limited in its deep-tissue imaging capabilities. In the future, advancements in microscopic technologies have the potential to unlock the fundamental biological mysteries of the plant cell, and thus provide profound insights into plant developmental biology.
Author contribution statement
MM and AOA wrote the paper. JVS supervised the work, provided critical suggestions and edited the paper.
References
Ascough GD, Novák O, Pěnčík A, Rolčík J, Strnad M, Erwin JE, Van Staden J (2009) Hormonal and cell division analyses in Watsonia lepida seedlings. J Plant Physiol 166:1497–1507
Bairu MW, Kane ME (2011) Physiological and developmental problems encountered by in vitro cultured plants. Plant Growth Regul 63:101–103
Bairu MW, Stirk WA, Van Staden J (2009) Factors contributing to in vitro shoot-tip necrosis and their physiological interactions. Plant Cell Tissue Organ Cult 98:239–248
Bairu MW, Aremu AO, Van Staden J (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63:147–173
Bird DA, Buruiana MM, Zhou Y, Fowke LC, Wang H (2007) Arabidopsis cyclin-dependent kinase inhibitors are nuclear-localized and show different localization patterns within the nucleoplasm. Plant Cell Rep 26:861–872
Blazquez S, Olmos E, Hernández JA, Fernández-García N, Fernández JA, Piqueras A (2009) Somatic embryogenesis in saffron (Crocus sativus L.). Histological differentiation and implication of some components of the antioxidant enzymatic system. Plant Cell Tissue Organ Cult 97:49–57
Boevink P, Oparka K, Cruz SS, Martin B, Betteridge A, Hawes C (1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15:441–447
Bouchabké-Coussa O, Obellianne M, Linderme D, Montes E, Maia-Grondard A, Vilaine F, Pannetier C (2013) Wuschel overexpression promotes somatic embryogenesis and induces organogenesis in cotton (Gossypium hirsutum L.) tissues cultured in vitro. Plant Cell Rep 32:675–686
Canhoto JM, Mesquita JF, Cruz GS (1996) Ultrastructural changes in cotyledons of pineapple guava (Myrtaceae) during somatic embryogenesis. Ann Bot 78:513–521
Canhoto JM, Rama SC, Cruz GS (2006) Somatic embryogenesis and plant regeneration in carob (Ceratonia siliqua L.). In Vitro Cell Dev Biol-Plant 42:514–519
Canter PH, Thomas H, Ernst E (2005) Bringing medicinal plants into cultivation: opportunities and challenges for biotechnology. Trends Biotechnol 23:180–185
Capelo AM, Silva S, Brito G, Santos C (2010) Somatic embryogenesis induction in leaves and petioles of a mature wild olive. Plant Cell Tissue Organ Cult 103:237–242
Caponetti JD, Gray DJ, Trigiano RN (2005) History of plant tissue and cell culture. In: Trigiano RN, Gray DJ (eds) Plant Development and Biotechnology. CRC Press, Florida, USA, pp 9–15
Carpentier AS, Abreu S, Trichet M, Satiat-Jeunemaitre B (2012) Microwaves and tea: new tools to process plant tissue for transmission electron microscopy. J Microsc 247:94–105
Chakrabarty D, Park SY, Ali MB, Shin KS, Paek KY (2006) Hyperhydricity in apple: ultrastructural and physiological aspects. Tree Physiol 26:377–388
Chalfie M, Kain SR (2005) Methods of biochemical analysis, green fluorescent protein: properties, applications and protocols, vol 47. Wiley-Interscience, New Jersey
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805
Chandler DE, Roberson RW (2009) Bioimaging: current concepts in light and electron microscopy. Jones and Bartlett Publishers, USA
Ckurshumova W, Caragea AE, Goldstein RS, Berleth T (2011) Glow in the dark: fluorescent proteins as cell and tissue-specific markers in plants. Mol Plant 4:794–804
Cole M, Jacobs B, Soubigou-Taconnat L, Balzergue S, Renou JP, Chandler JW, Werr W (2013) Live imaging of DORNRÖSCHEN and DORNRÖSCHEN-LIKE promoter activity reveals dynamic changes in cell identity at the microcallus surface of Arabidopsis embryonic suspensions. Plant Cell Rep 32:45–59
Correll MJ, Weathers PJ (2001) Effects of light, CO2 and humidity on carnation growth, hyperhydration and cuticular wax development in a mist reactor. In Vitro Cell Dev Biol-Plant 37:405–413
da Cruz ACF, Rocha DI, Iarema L, Ventrella MC, Costa MGC, Neto VBP, Otoni WC (2014) In vitro organogenesis from root culture segments of Bixa orellana L. (Bixaceae). In Vitro Cell Dev Biol-Plant 50:76–83
da Silva ML, Pinto DLP, Guerra MP, Floh ES, Bruckner CH, Otoni WC (2009) A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos. Plant Cell, Tissue Organ Cult 99:47–54
Dai J-L, Tan X, Zhan Y-G, Zhang Y-Q, Xiao S, Gao Y, Xu D-W, Wang T, Wang X-C, You X-L (2011) Rapid and repetitive plant regeneration of Aralia elata Seem. via somatic embryogenesis. Plant Cell, Tissue Organ Cult 104:125–130
Davidson MW, Campbell RE (2009) Engineered fluorescent proteins: innovations and applications. Nat Methods 6:713–717
De Boer HH, Van der Merwe AE, Maat GJR (2013) The diagnostic value of microscopy in dry bone palaeopathology: a review. Intl J Paleopathol 3:113–121
de Oliveira LM, Paiva R, de Santana JRF, Alves E, Nogueira RC, Pereira FD (2008) Effect of cytokinins on in vitro development of autotrophism and acclimatization of Annona glabra L. In Vitro Cell Dev Biol-Plant 44:128–135
Del Bianco M, Giustini L, Sabatini S (2013) Spatiotemporal changes in the role of cytokinin during root development. New Phytol 199:324–338
Demeter Z, Surányi G, Molnár VA, Sramkó G, Beyer D, Kónya Z, Vasas G, Hamvas M, Máthé C (2010) Somatic embryogenesis and regeneration from shoot primordia of Crocus heuffelianus. Plant Cell Tissue Organ Cult 100:349–353
Dewir Y, Singh N, Shaik S, Nicholas A (2010) Indirect regeneration of the Cancer bush (Sutherlandia frutescens L.) and detection of l-canavanine in in vitro plantlets using NMR. In Vitro Cell Dev Biol-Plant 46:41–46
Dobránszki J, Teixeira da Silva JA (2010) Micropropagation of apple—A review. Biotechnol Adv 28:462–488
Domozych DS (2012) The quest for four-dimensional imaging in plant cell biology: it’s just a matter of time. Ann Bot 110:461–474
El-Bakry M, Sheehan J (2014) Analysing cheese microstructure: a review of recent developments. J Food Eng 125:84–96
Fehér A, Pasternak TP, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tissue Organ Cult 74:201–228
Follet-Gueye M-L, Pagny S, Faye L, Gomord V, Driouich A (2003) An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco bright yellow (BY-2) cells. J Histochem Cytochem 51:931–940
Fontes MA, Otoni WC, Carolino SMB, Brommonschenkel SH, Fontes EPB, Fári M, Louro RP (1999) Hyperhydricity in pepper plants regenerated in vitro: involvement of BiP (Binding Protein) and ultrastructural aspects. Plant Cell Rep 19:81–87
George EF (1993) Plant propagation by tissue culture, part 1: the technology. Exegetics Ltd, London, UK
Grzebelus E, Szklarczyk M, Baranski R (2012) An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot. Plant Cell, Tissue Organ Cult 109:101–109
Guha S, Rao IU (2010) Culture surface and exogenous putrescine-altered shoot growth pattern in mannitol- and cadmium chloride-pretreated callus of Cymbidium Via del Playa “Yvonne”. In Vitro Cell Dev Biol-Plant 46:491–498
Gzyl J, Przymusiński R, Gwóźdź EA (2009) Ultrastructure analysis of cadmium-tolerant and -sensitive cell lines of cucumber (Cucumis sativus L.). Plant Cell, Tissue Organ Cult 99:227–232
Haseloff J, Siemering KR (2005) The uses of green fluorescent protein in plants. In: Green Fluorescent Protein. Wiley, pp 259–284. doi:10.1002/0471739499.ch12
Hashimoto T, Takahashi K, Sato M, Bandara PKGSS, Nabeta K (2011) Cloning and characterization of an allene oxide cyclase, PpAOC3, in Physcomitrella patens. Plant Growth Regul 65:239–245
Hazarika BN (2006) Morpho-physiological disorders in in vitro culture of plants. Sci Hortic 108:105–120
He Y, Guo X, Lu R, Niu B, Pasapula V, Hou P, Cai F, Xu Y, Chen F (2009) Changes in morphology and biochemical indices in browning callus derived from Jatropha curcas hypocotyls. Plant Cell Tissue Organ Cult 98:11–17
Holme IB, Brinch-Pedersen H, Lange M, Holm PB (2006) Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules. Plant Cell Rep 25:1325–1335
Hraška M, Rakouský S, Čurn V (2006) Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants. Plant Cell, Tissue Organ Cult 86:303–318
Huai J, Zheng J, Wang G (2009) Overexpression of a new Cys2/His2 zinc finger protein ZmZF1 from maize confers salt and drought tolerance in transgenic Arabidopsis. Plant Cell Tissue Organ Cult 99:117–124
Huang C-H, Chung J-P (2011) Efficient indirect induction of protocorm-like bodies and shoot proliferation using field-grown axillary buds of a Lycaste hybrid. Plant Cell Tissue Organ Cult 106:31–38
Iliev I, Kitin P (2011) Origin, morphology, and anatomy of fasciation in plants cultured in vivo and in vitro. Plant Growth Regul 63:115–129
Ivanova M, Van Staden J (2010) Natural ventilation effectively reduces hyperhydricity in shoot cultures of Aloe polyphylla Schönland ex Pillans. Plant Growth Regul 60:143–150
Jahn KA, Barton DA, Kobayashi K, Ratinac KR, Overall RL, Braet F (2012) Correlative microscopy: providing new understanding in the biomedical and plant sciences. Micron 43:565–582
Jausoro V, Llorente BE, Apóstolo NM (2010a) Structural differences between hyperhydric and normal in vitro shoots of Handroanthus impetiginosus (Mart. ex DC) Mattos (Bignoniaceae). Plant Cell Tissue Organ Cult 101:183–191
Jausoro V, Llorente BE, Apóstolo NM (2010b) Structural differences between hyperhydric and normal in vitro shoots of Handroanthus impetiginosus (Mart. ex DC) Mattos (Bignoniaceae). Plant Cell, Tissue Organ Cult 101:183–191
Juszczyk J, Krzywiecki M, Kruszka R, Bodzenta J (2013) Application of scanning thermal microscopy for investigation of thermal boundaries in multilayered photonic structures. Ultramicroscopy 135:95–98
Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188
Kajikawa K, Yamaguchi T, Katsuda S, Miwa A (1975) An improved electron stain for elastic fibers using tannic acid. J Electron Microsc 24:287–289
Kane ME (2005) Shoot culture procedures. In: Trigiano RN, Gray DJ (eds) Plant Development and Biotechnology. CRC Press, Washington D.C. USA
Kang YM, Park DJ, Min JY, Song HJ, Jeong MJ, Kim YD, Kang SM, Karigar CS, Choi MS (2011) Enhanced production of tropane alkaloids in transgenic Scopolia parviflora hairy root cultures over-expressing putrescine N-methyl transferase (PMT) and hyoscyamine-6β-hydroxylase (H6H). In Vitro Cell Dev Biol-Plant 47:516–524
Karuppusamy S (2009) A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. J Med Plant Res 3:1222–1239
Kozai T (1991) Photoautotrophic micropropagation. In Vitro Cell Dev Biol-Plant 27:47–51
Kozai T, Kubota C, Ryoung Jeong B (1997) Environmental control for the large-scale production of plants through in vitro techniques. Plant Cell Tissue Organ Cult 51:49–56
Kuo J (2007) Electron microscopy: Methods and protocols, vol 369. Humana Press Inc., New Jersey, USA
Kurczyńska EU, Gaj MD, Ujczak A, Mazur E (2007) Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh. Planta 226:619–628
Lai K, Yusoff K, Mahmood M (2013) Functional ectodomain of the hemagglutinin-neuraminidase protein is expressed in transgenic tobacco cells as a candidate vaccine against Newcastle disease virus. Plant Cell Tissue Organ Cult 112:117–121
Larkin P, Scowcroft W (1981) Somaclonal variation—A novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214
Lin G-Z, Zhao X-M, Hong S-K, Lian Y-J (2011) Somatic embryogenesis and shoot organogenesis in the medicinal plant Pulsatilla koreana Nakai. Plant Cell Tissue Organ Cult 106:93–103
Liu X, Sun L, Li C, Yang A, Zhang J (2013) Enhanced expression of the human CD14 protein in tobacco using a 22-kDa alpha-zein signal peptide. Plant Cell Tissue Organ Cult 112:9–18
López-Villalobos A, Dodds PF, Hornung R (2011) Lauric acid improves the growth of zygotic coconut (Cocos nucifera L.) embryos in vitro. Plant Cell Tissue Organ Cult 106:317–327
Mathur J (2007) The illuminated plant cell. Trends in Plant Sci 12:506–513
Mayer JLS, Stancato GC, Appezzato-Da-Glória B (2010) Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of Oncidium flexuosum Sims (Orchidaceae). Plant Cell Tissue Organ Cult 103:411–416
Mendes MD, Cristina Figueiredo A, Margarida Oliveira M, Trindade H (2013) Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius. Plant Cell Tissue Organ Cult 113:341–351
Mondal TK, Bhattacharya A, Laxmikumaran M, Singh Ahuja P (2004) Recent advances of tea (Camellia sinensis) biotechnology. Plant Cell Tissue Organ Cult 76:195–254
Motte H, Vereecke D, Geelen D, Werbrouck S (2014) The molecular path to in vitro shoot regeneration. Biotechnol Adv 32:107–121
Moyo M, Finnie JF, Van Staden J (2009) In vitro morphogenesis of organogenic nodules derived from Sclerocarya birrea subsp. caffra leaf explants. Plant Cell Tissue Organ Cult 98:273–280
Moyo M, Bairu MW, Amoo SO, Van Staden J (2011) Plant biotechnology in South Africa: micropropagation research endeavours, prospects and challenges. S Afr J Bot 77:996–1011
Moyo M, Finnie JF, Van Staden J (2012) Topolins in Pelargonium sidoides micropropagation: do the new brooms really sweep cleaner? Plant Cell Tissue Organ Cult 110:319–327
Moyo M, Koetle MJ, Van Staden J (2014) Photoperiod and plant growth regulator combinations influence growth and physiological responses in Pelargonium sidoides DC. In Vitro Cell Dev Biol-Plant 50:487–492
Muday GK, DeLong A (2001) Polar auxin transport: controlling where and how much. Trends in Plant Sci 6:535–542
Murphy DB, Davidson MW (2013) Fundamentals of light microscopy and electronic imaging, 2nd edn. Wiley-Blackwell, New Jersey, USA
Nakagawa R, Kurushima M, Matsui M, Nakamura R, Kubo T, Funada R (2011) Polyamines promote the development of embryonal-suspensor masses and the formation of somatic embryos in Picea glehnii. In Vitro Cell Dev Biol-Plant 47:480–487
Nebenführ A, Gallagher LA, Dunahay TG, Frohlick JA, Mazurkiewicz AM, Meehl JB, Staehelin LA (1999) Stop-and-Go movements of plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol 121:1127–1141
Neelakandan A, Wang K (2012) Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. Plant Cell Rep 31:597–620
Nogué F, Grandjean O, Craig S, Dennis S, Chaudhury M (2000) Higher levels of cell proliferation rate and cyclin CycD3 expression in the Arabidopsis amp1 mutant. Plant Growth Regul 32:275–283
Olmos E, Hellín E (1998) Ultrastructural differences of hyperhydric and normal leaves from regenerated carnation plants. Sci Hortic 75:91–101
Parra-Vega V, Renau-Morata B, Sifres A, Seguí-Simarro JM (2013) Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult 112:353–360
Pavlović S, Vinterhalter B, Zdravković-Korać S, Vinterhalter D, Zdravković J, Cvikić D, Mitić N (2013) Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis). Plant Cell Tissue Organ Cult 113:397–406
Pavoković D, Poljuha D, Horvatić A, Ljubešić N, Hagège D, Krsnik-Rasol M (2012) Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation. Plant Cell Tissue Organ Cult 108:111–119
Pence VC (2010) The possibilities and challenges of in vitro methods for plant conservation. Kew Bull 65:539–547
Picas L, Milhiet P-E, Hernández-Borrell J (2012) Atomic force microscopy: a versatile tool to probe the physical and chemical properties of supported membranes at the nanoscale. Chem Phys Lipids 165:845–860
Picoli EAT, Otoni WC, MrL Figueira, Carolino SMB, Almeida RS, Silva EAM, Carvalho CR, Fontes EPB (2001) Hyperhydricity in in vitro eggplant regenerated plants: structural characteristics and involvement of BiP (Binding Protein). Plant Sci 160:857–868
Pospíšilová J, Synková H, Haisel D, Semorádová S (2007) Acclimation of plantlets to ex vitro conditions: effects of air humidity, irradiance, CO2 concentration and abscisic acid (a review). Acta Hortic 748:29–38
Potocka I, Baldwin TC, Kurczynska EU (2012) Distribution of lipid transfer protein 1 (LTP1) epitopes associated with morphogenic events during somatic embryogenesis of Arabidopsis thaliana. Plant Cell Rep 31:2031–2045
Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229–233
Ptak A, Tahchy AE, Wyżgolik G, Henry M, Laurain-Mattar D (2010) Effects of ethylene on somatic embryogenesis and galanthamine content in Leucojum aestivum L. cultures. Plant Cell Tissue Organ Cult 102:61–67
Qi Y, Lou Q, Quan Y, Liu Y, Wang Y (2013) Flower-specific expression of the Phalaenopsis flavonoid 3′, 5′-hydroxylase modifies flower color pigmentation in Petunia and Lilium. Plant Cell Tissue Organ Cult 115:263–273
Quiala E, Cañal M-J, Meijón M, Rodríguez R, Chávez M, Valledor L, de Feria M, Barbón R (2012) Morphological and physiological responses of proliferating shoots of teak to temporary immersion and BA treatments. Plant Cell Tissue Organ Cult 109:223–234
Raju SC, Kathiravan K, Aslam A, Shajahan A (2013) An efficient regeneration system via somatic embryogenesis in mango ginger (Curcuma amada Roxb.). Plant Cell, Tissue Organ Cult 112:387–393
Ramachandra Rao S, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153
Ramakrishna A, Giridhar P, Ravishankar GA (2011) Calcium and calcium ionophore A23187 induce high-frequency somatic embryogenesis in cultured tissues of Coffea canephora P ex Fr. In Vitro Cell Dev Biol-Plant 47:667–673
Ramakrishna A, Giridhar P, Jobin M, Paulose CS, Ravishankar GA (2012) Indoleamines and calcium enhance somatic embryogenesis in Coffea canephora P ex Fr. Plant Cell, Tissue Organ Cult 108:267–278
Redha A, Suleman P (2011) Effects of exogenous application of polyamines on wheat anther cultures. Plant Cell Tissue Organ Cult 105:345–353
Reichel C, Mathur J, Eckes P, Langenkemper K, Koncz C, Schell J, Reiss B, Maas C (1996) Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells. Proc Natl Acad Sci USA 93:5888–5893
Roberts WS, Lonsdale DJ, Griffiths J, Higson SPJ (2007) Advances in the application of scanning electrochemical microscopy to bioanalytical systems. Biosens Bioelectron 23:301–318
Rocha DI, Vieira LM, Tanaka FAO, da Silva LC, Otoni WC (2012) Anatomical and ultrastructural analyses of in vitro organogenesis from root explants of commercial passion fruit (Passiflora edulis Sims). Plant Cell Tissue Organ Cult 111:69–78
Rojas-Martínez L, Visser RGF, de Klerk G-J (2010) The hyperhydricity syndrome: waterlogging of plant tissues as a major cause. Propag Ornam Plant 10:169–175
Rosa YBCJ, Dornelas MC (2012) In vitro plant regeneration and de novo differentiation of secretory trichomes in Passiflora foetida L. (Passifloraceae). Plant Cell Tissue Organ Cult 108:91–99
Rosa YBC, Aizza LCB, Armanhi JSL, Dornelas MC (2013) A Passiflora homolog of a D-type cyclin gene is differentially expressed in response to sucrose, auxin, and cytokinin. Plant Cell Tissue Organ Cult 115:233–242
Rout GR, Samantaray S, Das P (2000) In vitro manipulation and propagation of medicinal plants. Biotechnol Adv 18:91–120
Rout GR, Mohapatra A, Jain SM (2006) Tissue culture of ornamental pot plant: a critical review on present scenario and future prospects. Biotechnol Adv 24:531–560
Ruffoni B, Savona M (2013) Physiological and biochemical analysis of growth abnormalities associated with plant tissue culture. Hortic Environ Biotechnol 54:191–205
Rugkhla A, Jones MGK (1998) Somatic embryogenesis and plantlet formation in Santalum album and S. spicatum. J Exp Bot 49:563–571
Sáenz L, Azpeitia A, Chuc-Armendariz B, Chan JL, Verdeil JL, Hocher V, Oropeza C (2006) Morphological and histological changes during somatic embryo formation from coconut plumule explants. In Vitro Cell Dev Biol-Plant 42:19–25
Sato S, Adachi A, Sasaki Y, Ghazizadeh M (2008) Oolong tea extract as a substitute for uranyl acetate in staining of ultrathin sections. J Microsc 229:17–20
Sharifi G, Ebrahimzadeh H, Ghareyazie B, Karimi M (2010) Globular embryo-like structures and highly efficient thidiazuron-induced multiple shoot formation in saffron (Crocus sativus L.). In Vitro Cell Dev Biol-Plant 46:274–280
Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239
Shur J, Price R (2012) Advanced microscopy techniques to assess solid-state properties of inhalation medicines. Adv Drug Deliver Rev 64:369–382
Sirerol-Piquer MS, Cebrián-Silla A, Alfaro-Cervelló C, Gomez-Pinedo U, Soriano-Navarro M, Verdugo J-MG (2012) GFP immunogold staining, from light to electron microscopy, in mammalian cells. Micron 43:589–599
Sivanesan I, Song JY, Hwang SJ, Jeong BR (2011) Micropropagation of Cotoneaster wilsonii Nakai—a rare endemic ornamental plant. Plant Cell Tissue Organ Cult 105:55–63
Smith-Espinoza C, Bartels D, Phillips J (2007) Analysis of a LEA gene promoter via Agrobacterium-mediated transformation of the desiccation tolerant plant Lindernia brevidens. Plant Cell Rep 26:1681–1688
Smulders M, de Klerk G (2011) Epigenetics in plant tissue culture. Plant Growth Regul 63:137–146
Sreedhar RV, Venkatachalam L, Neelwarne B (2009) Hyperhydricity-related morphologic and biochemical changes in vanilla (Vanilla planifolia). J Plant Growth Regul 28:46–57
Steiner N, Santa-Catarina C, Guerra MP, Cutri L, Dornelas MC, Floh ES (2012) A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis. Plant Cell Tissue Organ Cult 109:41–50
Stone SL, Kwong LW, Yee KM, Pelletier J, Lepiniec L, Fischer RL, Goldberg RB, Harada JJ (2001) LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development. Proc Natl Acad Sci 98:11806–11811
Sun X, Ji W, Ding X, Bai X, Cai H, Yang S, Qian X, Sun M, Zhu Y (2013) GsVAMP72, a novel Glycine soja R-SNARE protein, is involved in regulating plant salt tolerance and ABA sensitivity. Plant Cell Tissue Organ Cult 113:199–215
Tang W, Newton RJ, Charles TM (2005) High efficiency inducible gene expression system based on activation of a chimeric transcription factor in transgenic pine. Plant Cell Rep 24:619–628
Teixeira da Silva JA (2003) Chrysanthemum: advances in tissue culture, cryopreservation, postharvest technology, genetics and transgenic biotechnology. Biotechnol Adv 21:715–766
Tewari RK, Prommer J, Watanabe M (2013) Endogenous nitric oxide generation in protoplast chloroplasts. Plant Cell Rep 32:31–44
Thomasson MS, Macnaughtan MA (2013) Microscopy basics and the study of actin–actin-binding protein interactions. Anal Biochem 443:156–165
Torrealba F, Carrasco MA (2004) A review on electron microscopy and neurotransmitter systems. Brain Res Rev 47:5–17
Tranfield EM, Walker DC (2013) The ultrastructure of animal atherosclerosis: what has been done, and the electron microscopy advancements that could help scientists answer new biological questions. Micron 46:1–11
Trigiano RN, Malueg KR, Pickens KA, Cheng Z-M, Graham ET (2005) Histological techniques. In: Trigiano RN, Gray DJ (eds) Plant Development and Biotechnology. CRC Press, Boca Raton, Florida, USA, pp 39–54
Valero-Aracama C, Kane M, Wilson S, Vu J, Anderson J, Philman N (2006) Photosynthetic and carbohydrate status of easy-and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes during in vitro culture and ex vitro acclimatization. In Vitro Cell Dev Biol-Plant 42:572–583
Van Eck J, Keen P (2009) Continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures. In Vitro Cell Dev Biol-Plant 45:750–757
Vasil I (2008) A history of plant biotechnology: from the cell theory of Schleiden and Schwann to biotech crops. Plant Cell Rep 27:1423–1440
Verpoorte R, Memelink J (2002) Engineering secondary metabolite production in plants. Curr Opin Biotech 13:181–187
Vieten A, Sauer M, Brewer PB, Friml J (2007) Molecular and cellular aspects of auxin-transport-mediated development. Trends Plant Sci 12:160–168
Wakte KV, Nadaf AB, Thengane RJ, Jawali N (2009) In vitro regenerating plantlets in Pandanus amaryllifolius Roxb. as a model system to study the development of lower epidermal papillae. In Vitro Cell Dev Biol-Plant 45:701–707
Werker E, Leshem B (1987) Structural changes during vitrification of carnation plantlets. Ann Bot 59:377–385
Whited AM, Park PSH (2014) Atomic force microscopy: a multifaceted tool to study membrane proteins and their interactions with ligands. Biochim Bioph Acta- Biomembranes 1838:56–68
Woo SM, Wetzstein HY (2008) Morphological and histological evaluations of in vitro regeneration in Elliottia racemosa leaf explants induced on media with thidiazuron. J Am Soc Hortic Sci 133:167–172
Wu Z, Chen LJ, Long YJ (2009) Analysis of ultrastructure and reactive oxygen species of hyperhydric garlic (Allium sativum L.) shoots. In Vitro Cell Dev Biol-Plant 45:483–490
Wu J-J, Liu Y-W, Sun M-X (2011) Improved and high throughput quantitative measurements of weak GFP expression in transgenic plant materials. Plant Cell Rep 30:1253–1260
Xu X, Guo R, Cheng C, Zhang H, Zhang Y, Wang X (2013) Overexpression of ALDH2B8, an aldehyde dehydrogenase gene from grapevine, sustains Arabidopsis growth upon salt stress and protects plants against oxidative stress. Plant Cell, Tissue Organ Cult 114:187–196
Yang Y, Yang L, Li Z (2013) Molecular cloning and identification of a putative tomato cationic amino acid transporter-2 gene that is highly expressed in stamens. Plant Cell, Tissue Organ Cult 112:55–63
Zacharias DA, Tsien RY (2005) Molecular biology and mutation of green fluorescent protein. In: Green Fluorescent Protein. Wiley, pp 83–120. doi:10.1002/0471739499.ch5
Zimmerman JL (1993) Somatic embryogenesis: a model for early development in higher plants. Plant Cell 5:1411–1423
Ziv M (1991) Vitrification: morphological and physiological disorders of in vitro plants. In: Debergh PC, Zimmerman RH (eds) Micropropagation: Technology and Applications. Kluwer Academic Publishers, Dordrecht, pp 45–69
Zumbusch A, Langbein W, Borri P (2013) Nonlinear vibrational microscopy applied to lipid biology. Prog Lipid Res 52:615–632
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The authors gratefully acknowledge financial support from Claude Leon Foundation, the University of KwaZulu-Natal and National Research Foundation, South Africa. We thank Dr W.A. Stirk for her valuable suggestions.
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Moyo, M., Aremu, A.O. & Van Staden, J. Insights into the multifaceted application of microscopic techniques in plant tissue culture systems. Planta 242, 773–790 (2015). https://doi.org/10.1007/s00425-015-2359-4
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DOI: https://doi.org/10.1007/s00425-015-2359-4