Ultrastructure study of Vanda Kasem’s Delight orchid’s protocorm-like body

Growing orchids has been classified as an international business since it covers 8% of the world floriculture trade. Thus, large-scale micropropagation of orchid using tissue culture techniques and improvement of some essential traits, such as resistances to various diseases and pests, and tolerances to environmental stresses, such as low temperatures and low light intensities, via genetic engineering acknowledged the orchids as one of the top ten cut flowers. Protocorm-like bodies (PLBs) are excellent explants for clonal propagation, artificial seeds development and genetic engineering since they are organized and easily regenerable somatic embryos that propagate rapidly. Vanda Kasem’s Delight hybrid orchid’s PLBs derived from shoot tip culture were examined in order to understand the fundamental ultrastructure of PLB. Examination of the cross sections of PLB revealed that PLB is made of a discrete bipolar structure that contains anterior and posterior meristems. Actively dividing meristem cells and outer layer lined by several rows of small and isodiametric cells with a dense cytoplasm and a prominent nucleus were also observed via cytological studies. Scanning Electron Microscope investigations revealed that the surface of PLBs is occupied by vertically positioned branched trichome appendages and regularly spaced stomatal openings with two guard cells. Organelles such as mitochondria of various sizes, shapes and biconvex chloroplast in the cytoplasm were revealed by Transmission Electron Microscope analysis. Hence, inheriting actively dividing cells, and bestowed with elements related to transpiration, photosynthesis and energy synthesizing power house makes PLB a suitable explant for micropropagation and genetic engineering studies.


Palavras
The genus Vanda comprises about T he development of the orchid industry is very rapid especially in Association of Southeast Asian Nations (ASEAN) such as Malaysia, Singapore, Indonesia and Thailand. Orchids are commercially important plants cultivated as cut flowers and and intense purplish blue coloration that may appear pinkish under sunlight. VKD is quite a pricy orchid variety due to the aesthetic attributes such as huge flat blossoms with rich and dark coloration. The attraction of orchid lovers towards VKD calls for the improvement of this breed in terms of longer shelf life than usual.
Hence, there is a necessity to examine ultra structure of VKD protocorm-like bodies (PLB) to analyse the suitability of it as a starting material for the trait improvement studies via genetic engineering. Therefore, the objective of the following study is to describe the ultrastructure of VKD orchid's PLBs via histological, scanning electron microscope (SEM) and transmission electron microscope (TEM) studies. It's the very first time of conducting ultrastructure studies in VKD PLBs.

MATERIAL AND METHODS
Plant material -Protocorm-like bodies (PLBs) of Vanda Kasem's Delight (VKD) initiated via shoot tip culture were subcultured from a long standing tissue culture by maintaining on Vacin & Went medium (1949) supplemented with 15% coconut water and 30% tomato extract. The pH (CyberScan PC 510 pH/mV/Conductivity/TDS/°C/°F Bench Meter, Eutech 73 Instruments, Singapore) of Vacin & Went medium in this study was adjusted to 4.8-5.0 prior to autoclaving (STURDY SA-300VFA-F-A505, Sturdy Industrial Co. Ltd., Taiwan). The culture was incubated at 25°C under 16 h photoperiod with cool white fluorescent light (supplied by Philips TLD fluorescent light tubes of 36 W, 150 μmol/m 2 /s). The PLBs were then subcultured every 4 weeks on modified Vacin & Went medium as mentioned previously to produce large quantities of explants for transformation. Healthy, greenish and rapidly growing PLBs were used for ultrastructure studies.
Histology procedure -Three 12week old PLBs were fixed in formalinacetic acid-ethanol (FAA) fixative solution for 24 hours. Fixed samples were washed under running tap water for another 24 hours. The PLBs were then dehydrated in a graded series of ethanol. On day 1, samples were dehydrated in 50% tert-butyl alcohol (TBA). On day 2, samples were first dehydrated with 70% ethanol for 7 hours followed  by 85% ethanol TBA. On day 3, 4, 5, and 6, samples were dehydrated for 24 hours in ethanol TBA 98%, ethanol TBA 100%, TBA I, and TBA II respectively. Dehydration was followed by clearance step (day 7) whereby the samples were immersed in xylene-substitute for 2 hours. During the wax absorption step samples were first immersed in a mixture of xylene-substitute and wax for 6 hours and then transferred to pure wax solution and left overnight. The following day, samples were treated twice with new wax solution and left overnight. Wax absorption was carried out in incubator at 60 o C. On day 9, melted wax was poured into mould to form wax blocks and the samples were immediately positioned inside the wax block. These blocks were then cut into thin slices under microtome cutter (Leica RM 2135) and the thin slices were allowed to rest on the clean glass slides. Glass slides containing sample slices were left in the incubator (Memmert, Germany) at 60 o C overnight. This was followed by staining procedure which was carried down in a transparent vertical glass jar with lining to hold the slides. Overnight dried slides were first dewaxed by rinsing in Histo-Clear solvent (National Diagnostics, USA) for 10 minutes. This was followed by rehydration of the slides in a series of reducing ethanol concentrations (100%, 90%, 70%, and 50%) for 2 min each. The slides were then stained with safranin for 20 min and rinsed in 70% and 80% ethanol for a minute respectively. This was followed by fast green staining for 2 mins. Care need to be taken as fast green tend to counter stain safranin as it's a strong stain. The slides were then dehydrated twice in 95% ethanol for a total of 2 minutes and finally rinsed in 100% ethanol for 2 min. The slides were then rinsed twice with Histo-Clear for a total of 6 min to wash the slides. The slides were then allowed to dry before mounting with cover slip using Shandon Histomount xylene substitute Mountant (Thermo Scientific). Stained slides were then viewed under light microscope (Olympus BX50, Olympus Optical Co. Ltd., Japan) fitted to a JVC K-F55B colour video camera (JVC Victor Company of Japan, Limited, Japan) and analysed with the Docu Version 3.1 image analysis system (Soft Imaging System, GmbH, Münster, Germany).

SEM tissue preparation -freeze drying technique -Three
PLBs were placed on a planchette on a damp filter paper lined Petri dish. The Petri dish was brought into fume hood and a few drops of 2% osmium tetroxide (OsO 4 ) were added to the filter paper. The Petri dish was closed immediately and kept in the fume hood for 2 hours. This is known as vapour fixation. Once the samples had been vapour fixed, they were plunged into liquid nitrogen and transferred on to the peltier-cooled stage of the freeze dryer for about 10 hours at varying temperature. The prepared samples were stored in a dessicator if not viewed immediately. The specimens were then sputter coated with 5-10 nm gold particles before viewing it using a Leo Supra 50VP Field Emission scanning electron microscope (Carl Zeiss SMT, Germany).

TEM tissue preparation -Three
PLBs were fixed in McDowell-Trump fixative at 4 o C for 24 hours. This fixative contains 4% formaldehyde and 1% glutaraldehyde in a 0.1 M phosphate buffer, and a pH of 7.2-7.4. Mix 86 mL distilled water, 10 mL Fisher F-79 37-40% formaldehyde, 4 mL 25% biological grade glutaraldehyde, 1.16 g NaH 2 PO 4 .H 2 O and 0.27 g NaOH in the stated order to prepare the McDowell-Trump fixative. The following day, the PLBs were washed in 0.1 M phosphate buffer three times at 10 minutes interval and post fixed in 1% osmium tetroxide prepared in the same buffer as above at room temperature for 2 hours. Then the PLBs were washed in distilled water twice at 10 minutes interval. The PLBs were dehydrated in a graded ethanol series [50% ethanol (15 minutes), 75% ethanol (15 minutes), 95% ethanol (15 minutes, twice), 100% ethanol (30 minutes, twice), and 100% acetone (10 minutes, twice)]. Resin containing a mixture of acetone and Spurr's resin was prepared at a ratio of 1:1. The resin-acetone mix was infiltrated into the samples in a rotator for 15-30 minutes followed by infiltration in Spurr's mix overnight in the rotator. Samples were infiltrated in a new change of Spurr's mix for another 5 hours in the rotator. Samples were embedded in polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Embedding was done by placing samples in a mould containing melted resin. Samples were cured at 60 o C for 12-48 h. Samples were then sliced using ultramicrotome and gathered on carbon planchette. Sliced ultrathin samples were stained with lead citrate and uranyl acetate and viewed under TEM.

RESULTS AND DISCUSSION
PLBs are composed of mass of  depression. Leroux et al. (1997) observed a similar depression on the protocorm of Cypripedium acaule which was caused by the displacement of the apical end. The depression determined the length of the apex and the scale. It is obvious in Figure 2a and b that the elongated scale folded over the apex located at the bottom of the depression on the adaxial side. The first true leaves on Figure 1d appeared after the apical dome subsided into the body of PLB.
SEM observation of the external surface of PLB revealed the regularly spaced stomatal openings (Figure 3a). They were anomocytic and possess two guard cells with no subsidiary cells (Figure 3b). Generally, care should be taken since cultured plants with nonfunctional stomata, weak root systems and poorly developed cuticles may cause mortality upon the transfer to ex vitro conditions (Mathur et al., 2008). Furthermore, high stomata density on VKD PLBs provides increased rates of stomatal conductance with improved transpiration efficiency.
Randomised projections of branched trichome on the surface of PLB were observed under SEM observation (Figure 4). Trichomes are bush-like appendages on the surface of plant tissues, which range in size from a few microns to several centimetres (Tissier,  Figure 1c). Contrarily, the inner cell layers are more voluminous than those of apical end. Inner layers are made of large cells with small nucleus and large vacuole (Figure 1c).
Examination of upper portion of PLB at higher magnification revealed a cap-like structure which covers the small anterior apical meristem ( Figure  1b). The cap-like structure is known as leaf primordial which eventually developed into first leaves (Figure 1d). Interestingly, SEM analysis of anterior meristematic pole of PLB showed elongated cone-shaped meristematic dome (Figure 2a and b). A more intense meristematic activity at one side of the dome displaced the apical end forming a hook-like structure depicting differentiated cells that are regarded as orchid embryos (Ng & Saleh, 2011) which develop with two discrete bipolar structures, where the upper region becomes the shoot while the bottom region becomes the root meristem (Jones, 2009). A notable anterior pole and posterior pole was observed on the PLB (Figure 1a). The bipolar nature of the 12-week old VKD PLB indicated the presence of apical meristems in VKD PLB (Figure 1a). Meristematic zone occupies 1/3 of the length of the PLB (Figure 1b). The bipolar PLB eventually differentiated anterior and posterior regions into shoot and root system respectively ( Figure 1d). Thus, meristematic PLB can be induced to regenerate into whole plantlets on plant growth regulator (PGR)-free medium (Ng & Saleh, 2011). Furthermore, the shoots develop directly from the primary and secondary meristems of PLB without an intervening explantorganogenesis phase (Ng & Saleh, 2011). Thus, absence of phytohormones and explant-organogenesis phase reduce the opportunity for somaclonal variation.
VKD PLB is made of cells that are actively involved in cell division. Cells with two nuclei beneath the epidermis layer were observed in PLB proving active mitosis (Figure 1c). In addition, Figure 1c shows the histochemical 2012). They can be single-celled or multicellular, curved or straight, nonsecretory or glandular, to name a few (Werker, 2000). Trichomes develop as projections from protodermal cells (Wagner, 1991). Trichomes are the very first structure to appear during differentiation of cultured cells (Wagner, 1991). Trichomes may serve as absorption or conduction tube since they are known to accommodate fluids inside (Gorb et al., 2007). Broken trichomes in Figure 4 show that trichomes are hollow in cavity which could be fluid filled.
A longitudinal sectioning of a PLB revealed the presence of non cytoplasmic inclusion, known as raphids ( Figure 5). Raphids are bundles of long needle-like (prismatic) calcium oxalate monocrystals and may occur in ordinary parenchymatous cells or special cells called idioblasts (Gomes & Lombardi, 2010). Raphids are mostly observed in young and actively growing tissues (Prychid & Rudall, 1999). They act as depositories of calcium ions and metabolic waste product (Sugimura et al., 1999) and play extracellular structural roles like fortifying the cell wall.
Transmission electron micrographs of the PLB revealed the presence of clusters of round shaped mitochondria with prominent cristae (highly folded inner membrane) and intercristal spaces (Figure 6a and b). TEM details of the PLBs cross section show chloroplast with the broad side of it parallel to the cell wall. Furthermore, plastoglobuli appearing as black spot was detected in the elongated chloroplast of PLBs ( Figure 7). Observations indicate that VKD PLBs possess biconvex chloroplast and the thylakoid are almost entirely filling the stroma of the plastid (Figure 7). Presence of mitochondria and chloroplast indicates that PLB is a differentiated organ that will function as an intact plant.
It could be concretely concluded that, bipolar VKD PLB is a transitory orchid embryo which proved to contain mass of differentiated cells that regenerated into an intact plantlet. VKD PLB is characterized by small isodiametric outer cells and voluminous inner cells. The presence of stomata, trichome, raphids, mitochondria and chloroplast indicates that physiological process of an intact plant is supported in the system of VKD PLB.