Microscopical analysis of in vitro Mokara Broga Giant orchid ’ s PLBs

Mokara orchids comes with variety of hybrids that have unique and highly variable types of characteristics such as petal colour, shape, size and other floral characteristics that make it very important economically and aesthetically. This study was conducted to evaluate on its histological and scanning electron microscopy analyses that make its PLBs to be a desirable biotechnological explant in propagation of this orchid as well as its importance in many biotechnological researches. Histological observation in Mokara Broga giant indicated the presence of meristematic tissues that are vital for biotechnological research such as genetic transformation, cryopreservation, micropropagation and others. It also indicated the presence of shoot primordia. Scanning electron microscopy analysis also indicated the developmental characteristics of PLBs in detail. Both analyses showed that the maturation of PLBs and gradual formation of shoot leaf primordial occurs through somatic embryogenesis and PLBs are vital explant in many plant tissue culture researches such as genetic transformation, cryopreservation, micropropagation and others.


Introduction
Orchidaceae, a family with 20,000 to 30,000 species and is the biggest family in the plant kingdom (Godo et al., 2010).Orchids are one of the most attractive clusters of ornamental plants and numerous novel cultivars have been formed by interspecific and intergeneric hybridization in order to produce plants with exotic and elegant flowers.Due environmental disruption, succession of natural habitats and overexploitation of horticultural, many orchid species are threatened today (Godo et al., 2010).
The most widespread use of orchids is as ornamental cut flowers.However, only few orchid plants are attractive enough for direct use as ornamentals.Orchids hybrids are used as ornamental due to they have beautiful foliage, shiny, multicoloured and decorated with prominent veins in elaborate patters.Orchids from several genera such as Mokara, Dendrobium, Anoectochilus, Goodyera, Ludisia (Haemaria) and Macodes are used widely.Orchids used indoor and outdoor landscapes are often employed to generate a special effect such as exotic surroundings, feeling of luxury and unique environment (Arditti, 1992;Dalayap et al., 2011).Generally, the flowers were made into corsages.In the orchid industry, trade in plants has always been and remains a significant aspect.However, in the past, this trade was limited to species, seedlings and matured plants.The establishment of clonal propagation procedures and hybridization of orchid such as Mokara hybrid has improved this market (Arditti, 1992;Dalayap et al., 2011).In return, it had brought a large numbers of outstanding cultivars which are mass-propagated by growers.Orchids are also used as a form of national flower such as Peristeriaelata in Panama (Arditti, 1992).
Mokara (Mokara spp.), commonly identified as "Smile Orchid" and it is native to Asia, where it has been first discovered and cultivated.It is a Vandaceous orchid resulting from a trigeneric hybridization between the Ascocentrum, Vanda and Arachnis orchids.The first Mokara hybrid was produced in Singapore in 1969 and was called Mokara Wai Liang, named after C.Y. Mok.Since then, several varieties were created where many of the hybrids have exclusive and highly variable starshaped flowers (Dalayap et al., 2011).This orchid has flowers with the largest number of colors compared to other orchids like purple, pink, blue, red, orange, yellow and coral with each and every colour has its own variety.The flower comprises of three sepals, which are usually diverse from the petals in shape but not in colour (Dalayap et al., 2011).There are also three petals but one has been significantly altered into a lip or labellum, which is a very complex structure and quite different from the other two sepals (Soon, 1989;Dalayap et al., 2011).This orchid group is therefore vital economically due to these unique floral characteristic.
In vitro propagation is the utmost popular method used for the multiplication of orchids as this method is found to be more effective than sexual propagation and other vegetative procedures (Talukder et al., 2003).However, tissue culture in orchids via indirect method which produce callus is not favoured due to lower growth rate and regular occurrence of necrosis in culture (Zhao et al., 2008).According to Chugh et al. (2009), the future of orchid propagation is most expected through multiplication of true-to-type plants through in vitro techniques.Several methods of propagation have been developed for orchids through in vitro culture of various parts including shoot tips, flower stalk nodes, buds, root tips and rhizome segments (Chen et al., 2000;Park et al., 2002b) with varying responses.
The most commonly used micropropagation techniques include direct and indirect shoot organogenesis and somatic embryogenesis.Somatic embryogenesis is more necessary biotechnological technique for large-scale clonal propagation of elite genotypes, development of synthetic seed technology, plant transformation (Bomal and Tremblay, 2000;Bandyopadhyay and Hamill, 2000) and conservation of threatened plant species.The most ideal regeneration systems for genetic transformation are direct and repetitive production of somatic embryos or de novo shoot organogenesis originating from single cells in the epidermis (Rugkhla and Jones, 1998).Whereas, genetic clones created through shoot organogenesis require further manipulation for root initiation, in somatic embryogenesis complete plants with a bipolar axis, vascular system and functional meristems are created in a single step (Bassuner et al., 2007).In somatic embryogenesis, somatic cells develop into plants in similar developmental stages to zygotic embryogenesis (Gomes et al., 2006).
Recently, numerous reports have described shoot organogenesis through somatic embryo-like structures from different explants, mainly leaves and hypocotyls.Histological observations of the embryo-like structures have established the initial globular-shaped morphological features (Haensch, 2004;Tian et al., 2008) during the early developmental stages of their growth, in synchrony with conventional somatic embryogenesis.These embryo-like structures have been mistaken for true somatic embryos (Salaj et al., 2005).Our present study describes the histological approach to determine the anatomical characteristics of somatic cells within the PLBs.To the best of our knowledge this is the first report on the histological analysis in Mokara Broga Giant.
Therefore, this study aims to evaluate the importance of the structural conditions by histological analysis and scanning electron microscope analysis of this orchid PLBs that confirms its structure as somatic embryos that makes it vital in the orchid propagation industry.

Histological Analysis Fixation
Mokara Broga Giant PLBs were fixed in FAA (95% ethyl alcohol: glacial acetic acid: formaldehyde: water, 10:1:2:7) for 1 week.Fixation is a process whereby the biological tissues are preserved from decay, stops the biochemical reactions and also increase the mechanical strength or stability of the treated tissues.

Dehydration
The PLBs were rinsed with distilled water a period of 1 hour with every 15 minutes of rinsing.PLBs were then transferred into alcohol tertiary-butyl alcohol, TBA which was done with varying concentration of (TBA) (50-100%); 50% TBA for 2 hours, 70% TBA for 2 hours, 85% TBA for 2 hours, 98% TBA for 2 hours and 100% TBA for 3 hours.Following that, the PLBs were treated with TBA I and TBA II for 3 hours respectively, and then treated PLBs were left overnight.

Clearing
PLBs were subsequently exposed to xylene for a period of 10 minutes followed by treatment xylene and wax for 30 minutes and subsequently samples were treated with wax I, II, and III (xylene and Shandon Histoplast Pelletised Paraffin Wax, Thermo Scientific) for 1 hour respectively at 60°C in an oven (Memmert, Germany).

Blocking, slicing and staining of samples
PLBs samples were then blocked and sliced using 6 Micron Microtome (Leica RM 2135).Then, the samples were then stained with safranin and fast green.Safranin, a basic dye which stains acidic features of tissue such as nuclei and lignified secondary cell walls that surround mature xylem cells in vascular bundles.Fast Green, an acidic dye that stains the basic components of tissue such as the cytoplasm and cellulosic primary cell walls.Slides were observed by light microscope (Olympus BX41).

Scanning Electron Microscope Analysis
PLBs samples were processed by freeze drying technique and the specimens were observed using Scanning Electron Microscope (Leo Supra 55VP-Ultra High Resolution analytical FESEM).

Results and Discussion
Propagation of orchids by the development of secondary PLBs from protocorms or PLBs has been defined for several orchids including Phalaenopsis (Chen and Chang, 2004;Murdad et al., 2006), Dendrobium (Saiprasad et al., 2004), Aerides (Sheelavanthmath et al., 2005) and Cymbidium (Teixeira da Silva and Tanaka, 2006).Numerous comparative analyses of plant responses to various types of plant growth regulators and the addition of organic additives to the culture medium to promote in vitro growth and proliferation of orchid is a common practice (Ichihashi and Islam, 1999;Chai et al., 2002;Islam et al., 2003;Rahman et al., 2004;Arditti, 2008, George et al., 2008).
The use of organic additives and PGRs may add toward the development of a simple and economical plant culture (Islam et al., 2003).Numerous comparative analyses of plant responses to diverse types of cytokinin (BA and kinetin) have revealed that the presence of BA in the culture medium produces higher frequencies of induction and proliferation of PLB in some orchids, such as Dendrobium nobile, Dendrobium Densiflorum, and Cymbidium aloifolium (Nayak et al., 2002;Sheelavanthmath et al., 2005;Luo et al., 2008).However, Teixeira da Silva and Tanaka ( 2006) specified that kinetin was more effective in promoting the formation of PLB in Cymbidium hybrids when supplemented together with NAA to the culture medium.In contrast, Chen et al. (2002) described that both BA and kinetin were equally effective for PLB proliferation in Epidendrumradicans.
Instead, Chai et al. (2002) described that the addition of a suitable amount of organic additives to the culture media significantly promoted the growth of PLBs in Phalaenopsis.This review displayed that varying conditions are desirable for diverse types of orchids to form their optimum PLBs growth.The presence of meristematic cells in PLBs can develop from PLBs wound surface, maturing into shoot primordial and can be converted into plantlets through somatic embryogenesis (Figure 1).The histological observation showed that the protocorm-like body of Mokara Broga Giant orchid comprises of the shoot apical meristem (SAM) and leaf primodia of sheath leaves at the interior region and larger cells act as the storage area at the posterior region (Figure 2).Histological observation presented the presence of an area having dense cytoplasm located on the anterior side of the PLB, showing meristematic cells, embedded below it with parenchymatous tissue with thinwalled cells.This parenchymatic mass of cells protruded from the surface on the upper part of the protocorm and associated with vascular tissue (Figure 3).
Furthermore, dividing cells of the SAM were smaller in size and were more densely organized (Figures 2, 3).Further development of these cells gave SAM a shape of a dome.The shoot primordial (SP) was also noted and is different.They comprised of small cells, undergoing anticlinal and periclinal divisions, still surrounded by large, apparently isodiametric parenchyma cells (Figures 2, 3).SEM study observation using freeze drying method displayed the presence of enlarged globular PLBs shape which consists of epidermal cells with rough surface texture, which gradually tend to become rougher and wrinkled at the further developmental stages (4 weeks old) (Figure 3).The embryo axis also displayed the development of LP after formation of a constriction (C) at the top of the globular head (Figure 4).There are more than one constriction (C) can be detected on a single PLB head (Figure 4).The leaf primordial (LP) was also noted (Figures 4, 5).Even though wide research has been carried out on in vitro PLB in cultivated orchids, there are limited complete structural details known on the histological observation of PLBs (Park et al., 2002a).
In the histological analysis, we presented that the presence of parenchyma cells next to the meristematic cells.The parenchyma tissue close to the core played an evident storage function (Sgarbi et al., 2009).The histological and SEM studies carried out in the present work displays that the meristematic core was enveloped by a mass of parenchymatic cells.The existence of meristematic tissue at the leaf apex discloses a characteristic that is inherent to the Orchidaceae family (Churchill et al., 1973).This study offered the morphological development observed with naked eye is substantiated with the details based on histological as well as SEM observations.Haensch (2004) described that Pelargonium hortorum observed that although globular and heart-shaped embryo-like regenerants were formed, histological analysis revealed that they lacked a defined root pole.In contrast to somatic embryogenesis where the globular-stage usually comprises of small cells with dense cytoplasm and large nuclei (Haensch, 2004), globular embryo-like structures had vacuolized parenchymatous cells (Salaj et al., 2005).Similar structures observed in other plant species have also been stated to as meristemoids, promeristems, meristemoid-like precursors (Hicks, 1994), protocorm-like bodies (Young et al., 2000;Tian et al., 2008), nodules (Batista et al., 2000;Xie and Hong, 2001;Ferreira et al., 2009) and nodular meristemoids (McCown et al., 1988).McCown et al. (1988) describe nodules as independent, spherical, dense cell clusters which are cohesively bound together and display consistent internal cell or tissue differentiation and loosely resemble protocorms.In contrast, a meristemoid can be described as a cluster of cells acting together as a meristematic centre (McCown et al., 1988).Hence, a histological approach is vital in distinguishing true somatic embryos and nodular meristemoids.Many published reports on somatic embryogenesis have drawn conclusions on the basis of morphological appearance only and should be reviewed critically (Bassuner et al., 2007).Histological approaches can offer critical information to allow the application of the most suitable in vitro plant regeneration methods (Woo and Wetzstein, 2008).
Histological and SEM observations through the presence of meristematic cells clearly presented that PLBs can develop from explants wound surface and can be converted into plantlets through sequential organogenesis or somatic embryogenesis.Since the PLBs comprised of multiple meristematic centres, it can be differentiated into shoot, leaf and then into plantlets gradually.Tian et al. (2008) reported that differentiation of globular cells led to formation of meristematic centers, which developed into PLBs.The SEM observations also definite that PLBs clearly exhibit shoot primordial and a meristem dome.The formation of PLBs is a unique characteristic of Orchidaceae and the term specifies a structure from induction of globular swellings until SP occur, without a root apparatus (Batygina et al., 2003).Therefore, due to the existence of abundant meristematic tissues in PLBs, propagation of orchids using tissue culture techniques has been practiced for more than a century and has resulted in the production of uniform clones in many orchid genera.The formation of protocorms from germinated seed and the successive induction of protocorm-like bodies (PLBs) or callus from the protocorm, stem-node, shoot-tip, leaf, root-tip, or root-tuber explants has become a reliable technique for propagating orchids (Park et al., 2003;Anjum et al., 2006;Hong et al., 2008;Medina et al., 2009).Propagation through PLB formation is preferred by commercial growers of most orchid genera due to the large number of PLBs that can be obtained within a moderately short period of time.The largescale propagation of PLBs can also be accomplished using a bioreactor system (Park et al., 2000).PLBs are also the common target tissue for genetic transformation studies in orchids since they can proliferate rapidly and have high abilities to regenerate into complete plantlets (Liau et al., 2003;Sreeramanan et al., 2008).Furthermore, PLBs can also serve as an appropriate plant material for cryopreservation experiment (Yin and Hong, 2009).PLBs are well-differentiated tissues that are sometimes viewed as orchid embryos that develop with two discrete bipolar structures, namely, the shoot and root meristem.Consequently, these structures are capable to convert to plantlets certainly when grown on plant growth regulator (PGR)-free medium.Besides, the PLBs directly formed from meristem tissue will display an advance genetic stability than those produced by callus (Luo et al., 2008;Gantait and Uma Rani, 2012).

Conclusion
Therefore, the presence of abundant meristematic cells within the PLBs displayed importance in orchid propagation through orchid tissue culture which makes it to be a prospective explant for cryopreservation and genetic transformation studies.