Morphology, flow cytometry and molecular assessment of ex-vitro grown micropropagated anthurium in comparison with seed germinated plants

Micropropagated and seed propagated plantlets of anthurium (Anthurium andreanum Lind. cv. CanCan) were transferred to similar field condition and growth stage. A comparative study was conducted using morphological parameters, ploidy level assessment as well as deoxyribonucleic acid (DNA) fingerprinting using inter simple sequence repeat (ISSR) markers. The in vitro generated anthurium plants exhibited comparable vegetative growth and more sucker production when compared to plants propagated through seeds. No variation in ploidy level was established through flow cytometric study. Genetic assessment through ISSR showed no polymorphism in banding pattern. It was revealed that there was no significant variation between micropropagated and seed propagated plants at ploidy and molecular level assuring the trueness of the micropropagated anthurium clones and their commercial applicability.Key words: Anthurium andreanum Lind, Ex vitro performance, ISSR, morphological competence, ploidy level

Detection of somaclonal variation by random amplified polymorphic DNA analysis during micropropagation of Phalaenopsis bellina (Rchb.f.) Christenson

Phalaenopsis bellina (Rchb.f.) Christenson orchid species are known for their beautiful flower shape, graceful inflorescence and fragrance. Protocorm-like bodies (PLBs) of P. bellina were induced from leaf segments. The PLBs were then subjected to proliferation using ½ strength Murashige and Skoog (MS) media with two subcultures at three months intervals. Twelve decamer random amplified polymorphic DNA (RAPD) primers were used to study somaclonal variation among the mother plant, the initially induced PLBs and proliferated PLBs after 3 and 6 months in culture. Eight out of twelve primers produced 172 bands with 18 polymorphic bands in all the treatments. The amplified products varied between 125 to 8000 bp. Among the primers used, P 16 produced the highest number of bands (29), while primer OPU 10 produced the lowest number (15). The range of similarity coefficient was from 0.83 to 1.0 among the different sub-cultures and mother plant (MP). It was found that minimal or no changes occurred between the MP and the PLBs produced after 3 months of induction. The induced PLBs were then subcultured for six months for proliferation and this resulted in about 17% dissimilarity with MP. It is reported that micropropagation of P. bellina can be carried out successfully using ½ strength MS media for 6 months but further proliferation may result in somaclonal variation which might change the prolific characteristic of this orchids.Key word: Moth orchid, somaclonal variation, random amplified polymorphic DNA, protocorm-like bodies

Effect of plant growth regulators and activated charcoal on in vitro growth and development of oil palm (Elaeis guineensis Jacq. var. Dura) zygotic embryo

The effect of plant growth regulators and activated charcoal (AC) on in vitro regeneration and plantlet development of oil palm (Elaeis guineensis Jacq. var. Dura) zygotic embryos were assessed. Zygotic embryos were cultured on Murashige and Skoog (MS) medium supplemented with a blend of 0.05 or 0.1 mg/L of each plant growth regulators (PGR) (gibberellic acid, 6-benzlaminopurine and α-naphthaleneacetic acid) without or with 2 g/L AC. The growth and development of the embryos were affected by the types of media formulations. Zygotic embryos cultured on MS medium supplemented with both PGR and AC enhanced shoot initiation and subsequent plantlet development, while PGR supplemented MS media without AC led to abnormal growth, suggesting that AC is indispensable for oil palm plantlet regeneration in vitro. The best medium for growth and development of plantlets was MS medium supplemented with 0.1 mg/L PGR and 2 g/L AC which significantly increased plantlet height (9.4 cm) as well as root length (4.4 cm) than the remaining media formulations.Key words: Activated charcoal, oil palm, plant growth regulators, zygotic embryo

Preliminary analysis of cryopreservation of Dendrobium Bobby Messina orchid using an encapsulation-dehydration technique with Evans blue assay

In vitro grown protocorm-like bodies (PLBs) of Dendrobium Bobby Messina hybrid were cryopreserved in liquid nitrogen (LN) at -196°C by an encapsulation-dehydration technique. PLBs (1 to 2 and 3 to 4 mm) were precultured in half strength semi-solid MS media supplemented with six different concentrations of sucrose (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 M). The PLBs were then encapsulated to form the beads in halfstrength liquid MS media supplemented with different concentrations of sodium alginate (2.5, 3.0 and 3.5%). The beads were placed in 2 ml cryovials and plunged into LN for 24 h. The beads were then thawed in a 40°C water bath for 90 s and were placed in recovery media composed of half strength semisolid MS media supplemented with 2% sucrose for four days under dark condition. After 12 days, the Evans blue dye assay was carried out to determine the viability of the PLBs. The highest viability was found in 1 to 2mm PLBs precultured in half strength semi-solid MS media supplemented with 1.0 M sucrose and encapsulated in 2.5% sodium alginate. Biochemical content analyses (chlorophyll, total soluble protein and peroxidase activities) were done to investigate the physiological responses of the PLBs after cryopreservation.Key words: Orchid, protocorm-like bodies, Dendrobium Bobby Messina, encapsulation-dehydration

Fundamental concept of cryopreservation using Dendrobium sonia-17 protocorm-like bodies by encapsulation-dehydration technique

This study was carried out to evaluate the potential of using the encapsulation-dehydration technique on cryopreservation protocorm-like bodies (PLBs) of Dendrobium sonia-17. The survival of the PLBs was assessed based on the effects of 4 dehydration periods (0, 1, 3 and 5 h) and 4 different concentrations of 24-h sucrose pretreatment (0, 0.3, 0.5 and 0.7 M). Upon dehydration, moisture content was determined and the PLBs were evaluated for survival using absorbance values from 2, 3,5- triphenyltetrazolium chloride (TTC) assay at 530 nm and regeneration observations. Moisture content declined with the dehydration time, but the decline was not significant for encapsulated PLBs. All cryopreserved PLBs gave very low survival irrespective of the dehydration period. The best survival percentage in the cryopreservation of the PLBs of Dendrobium sonia-17 was obtained when the combination of 0.5 M sucrose pretreatment and 3 h dehydration time was applied in the experiment.Key words: Dendrobium sonia-17, protocorm-like bodies, encapsulation-dehydration, 2,3,5-triphenyltetrazolium chloride (TTC) assay

Occurrence of glucosylsucrose [α-D-glucopyranosyl- (1→2)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside] and glucosylated homologues in cyanobacteria: Structural properties, cellular contents and possible function as thermoprotectants

Little is known about the structure and function of oligosaccharides in cyanobacteria. In this study, a new class of saccharides from Nostoc was identified by MS and NMR techniques, consisting of α-d-glucopyranosyl-(1→2)-[α-d-glucopyranosyl-(1→2)]n-β-d-fructofuranosides ranging from the trisaccharide (n = 1) to decasaccharide (n = 8). In Nostoc ellipsosporum the cell content of saccharides increased 10–20-fold after heat stress (1 day, 40 °C) or during prolonged cultivation. Under these conditions the abundance of homologues of higher molecular mass (> pentasaccharide) increased and finally exceeded that of homologues of lower molecular mass including sucrose. Total intracellular content of the saccharides after heat stress was 5–10 mg·(g dry weight)-1 corresponding to intracellular concentrations of 0.25–0.5% (w/v). A possible role of the oligosaccharides identified is in the protection of enzymes against heat inactivation. Whereas amylase from Nostoc was only weakly protected by the decasaccharide, α-amylase from porcine pancreas was more efficiently stabilized by the octasaccharide and decasaccharide. Evidence is presented for the widespread occurrence of the newly identified saccharides in cyanobacteria. The results are discussed including previous reports on cyanobacterial oligosaccharides and with respect to possible functions of these compounds in the living cell