The Development Of Encapsulation-Dehydration And Vitrification Protocols For Protocorm-Like Bodies (PLBs) Of Dendrobium Sonia-28

The vitrification and encapsulation-dehydration methods of cryopreservation were applied on protocorm-like bodies (PLBs) of the orchid hybrid Dendrobium sonia-28, with survival assessments conducted through growth observations, visualand spectrophotometric 2,3,5-triphenyltetrazolium chloride (TTC) assays. The best survival (16.0% regeneration) for vitrification was obtained when 3-4mm PLBs were precultured in semi-solid half-strength MS medium supplemented with 0.4M sucrose, loaded in a loading solution for 20 minutes, dehydrated for 50 minutes at 0°C in plant vitrification solution 2 (PVS2), cryopreserved in liquid nitrogen (LN) for 24 hours, thawed in a 40±2°C water bath for 90 seconds, unloaded in 1.2M sucrose for 20 minutes, and regenerated in semi-solid half-strength MS medium containing 2g.L-1 charcoal, with all media supplemented with 0.6mM ascorbic acid

Cryopreservation of PLBs of Brassidium Fly Away Using Encapsulation-Dehydration Techniqu

In vitro grown protocorm-like bodies (PLBs) of Brassidium Fly Away orchid hybrid were cryopreserved using encapsulation- dehydration technique. The viability of the cryopreserved cells was determined by 2,3,5-triphenyltetrazolium chloride (TTC) assay. For the preculture treatment, the PLBs were excised into two standard sizes of 1-2 and 4-5 mm and were precultured on half-strength Murashige and Skoog (MS) semi solid medium supplemented with diff erent concentrations of sucrose (0, 0.2, 0.4, 0.6, 0.8 and 1.0M). The PLBs size 4-5 mm and 0.6 M sucrose concentration was selected based on highest viability obtained in TTC assay. The PLBs were encapsulated for 30 minutes using 3% (w/v) liquid sodium alginate medium supplemented with 0.4M sucrose and 0.1M calcium chloride and osmoprotected in 0.75M sucrose solution for 24 hours at 25°C. The beads were then dehydrated using 50g heat-sterilised silica gel for four hours, cryopreserved for 24 hours, thawed in a 40±2°C water bath for 90 seconds, and regenerated in semi-solid half-strength. Biochemical analyses were conducted and the cryopreserved PLBs had produced lower content of chlorophyll while the highest specifi c peroxidase activity was observed in cryopreserved PLBs

Effect of PVS2 vitrification on Brassidium shooting star orchid using protocorm-like bodies (PLBs)

A cryopreservation procedure was developed to preserve protocorm-like bodies (PLBs) of Brassidium Shooting Star using the PVS2 vitrification technique. The optimised protocol involved the preculture of 3-4mm PLBs in half-strength Murashige and Skoog (MS) semi-solid medium supplemented with 0.8M sucrose, followed by dehydration in PVS2 solution for 20 minutes at 0°C, prior to storage in liquid nitrogen. The viability of non-cryopreserved and cryopreserved PLBs was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) assay, after two weeks of recovery. The chlorophyll contents, total soluble protein and peroxidase activities of both non-cryopreserved and cryopreserved PLBs were assayed after three weeks of recovery. The results from the biochemical analyses indicated that control PLBs produced the highest viability, followed by treatment on non-cryopreserved PLBs (-LN) and cryostored PLBs (+LN), except in the peroxidase activity assay. The peroxidase activity was detected as the highest in cryostored PLBs followed by treated but non-cryopreserved PLBs, and control PLBs

An improved PVS2 cryopreservation technique for Ascocenda Wangsa Gold orchid using protocorm-like bodies

Ascocenda Wangsa Gold is a new and fascinating orchid hybrid in the Malaysian flower industry. An efficient plant vitrification solution 2 (PVS2) cryopreservation technique was developed for protocorm-like bodies (PLBs) of Ascocenda Wangsa Gold orchid. Parameters assessed included the effect of PVS2 exposure periods, thawing duration, temperature, and culture conditions based on 2,3,5-triphenyltetrazolium chloride absorbance readings and regrowth rates. A regrowth rate of 33.3% was obtained after 2 months when the PLBs were dehydrated in PVS2 for 30 min. The growth rate was improved to 47% when thawing was conducted at 45 °C for 85 s. The highest growth rate (53.3%) was obtained when the PLBs were subjected to a 7-day dark treatment before being transferred to a 16-h/8-h light/dark photoperiod. Histological analyses were conducted to study the morphology of cryopreserved and noncryopreserved PLBs of Ascocenda Wangsa Gold. The vitrification protocol developed in this study is a feasible and safe method for strengthening the germplasm conservation of this orchid for commercial purposes

Catalase and superoxide dismutase activities and the total protein content of protocorm-like bodies of Dendrobium sonia-28 subjected to vitrification

Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, the genus faces both low germination rates and the risk of producing heterozygous progenies. Cryopreservation is currently the favoured long-term storage method for orchids with propagation problems. Vitrification, a frequently used cryopreservation technique, involves the application of pretreatments and cryoprotectants to protect and recover explants during and after storage in liquid nitrogen. However, cryopreservation may cause osmotic injuries and toxicity to cryopreserved explants from the use of highly concentrated additives, and cellular injuries from thawing, devitrification and ice formation. Reactive oxygen species (ROS), occurring during dehydration and cryopreservation, may also cause membrane damage. Plants possess efficient antioxidant systems such as the superoxide dismutase (SOD) and catalase (CAT) enzymes to scavenge ROS during low temperature stress. In this study, protocorm-like bodies (PLBs) of Dendrobium sonia-28 were assayed for the total protein content, and both SOD and CAT activities, at each stage of a vitrification exercise to observe for deleterious stages in the protocol. The results indicated that cryopreserved PLBs of Dendrobium sonia-28 underwent excessive post-thawing oxidative stress due to decreased levels of the CAT enzyme at the post-thawing recovery stage, which contributed to the poor survival rates of the cryopreserved PLBs

Molecular stability of protocorm-like bodies of Dendrobium sonia-28 after encapsulation- dehydration and vitrification

Abstract Dendrobium sonia-28 is prized for its pink-coloured and good cut flowers. Cryopreservation, requiring little space and maintenance, is touted as an important tool for long-term storage of plant genetic resources. The vitrification and encapsulation-dehydration methods of cryopreservation were applied on protocorm-like bodies (PLBs) of the orchid hybrid Dendrobium sonia-28, with survival and stability assessments conducted through growth observations and RAPD analysis respectively. Results obtained from both the control encapsulation-dehydration (53.3%) and vitrification (30.0%) experiments indicated that the encapsulation-dehydration treatment is less damaging to non-cryopreserved PLBs. However, the vitrification treatment produced PLBs that survived cryopreservation (16.0%). No surviving PLBs were obtained from the encapsulation-dehydration experiment. The RAPD analyses of PLBs of Dendrobium sonia-28 indicated that PLBs that were vitrified and cryopreserved were genetically stable, while those that were encapsulated, dehydrated and then cryopreserved were not genetically stable when compared to the stock culture

Refinement of a vitrification protocol for protocorm-like bodies of Dendrobium sonia-28.

The hybrid Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, it faces the risk of producing heterozygous progenies through conventional seed propagation or somaclonal variation. Cryopreservation is a favoured long-term storage method in the preservation and maintenance of orchids with specific desired traits. This paper presents a successful cryopreservation protocol for protocorm-like bodies (PLBs) of Dendrobium sonia-28 through the vitrification technique and the use of ascorbic acid and charcoal. Survival assessments were conducted through growth evaluations and visual and spectrophotometric 2,3,5-triphenyltetrazolium chloride assays. Results of this study, an improvement over the previous protocol applied to this orchid hybrid, showed that the addition of ascorbic acid to the preculture, loading, dehydration, unloading, and regeneration media, coupled with the addition of charcoal to the regeneration medium, helped boost recovery rates of cryopreserved PLBs of Dendrobium sonia-28

Research Note Polymorphism analysis of cryopreserved Dendrobium Bobby Messina protocorm-like bodies (PLBs) using RAPD markers

Abstract Dendrobium Bobby Messina, a sympodial orchid, is a new Dendrobium orchid hybrid from Indonesia with the parentage of (Imelda Romualdez × Jaquelyn Thomas). This orchid is cultivated for its cut flowers and potted plants, and is valued for its attractive flowers. Cryopreservation is a feasible long term storage method, whereby plants are stored at ultra low temperature using liquid nitrogen (-196⁰C). Protocorm-like bodies (PLBs) are an attractive target explant for orchid cryopreservation. In this study, cryopreservation using plant vitrification solution 2 (PVS2) was carried out on PLBs of Dendrobium Bobby Messina using optimum conditions, and growth recovery was obtained at 40%. In the screening of suitable primers to detect polymorphism in both cryopreserved and noncryopreserved PLBs in comparison with the stock culture, 20 primers were used but only 10 primers were able to generate bands. However, the number of bands produced in cryopreserved and non-cryopreserved PLBs from 3 primers and 1 primer indicated polymorphism and partial polymorphism respectively. The RAPD results from 6 selected primers indicated that the genetic stability of PLBs following cryopreservation was maintained. Hence, this method can be utilised as a potential cryopreservation approach for the germplasm conservation of Dendrobium Bobby Messina orchid

Vitrification and histological analyses of protocorm-like bodies of Vanda Kaseem's Delight orchid

Abstract This study was conducted to investigate the potential of cryopreserving protocorm-like bodies (PLBs) of Vanda Kaseem's Delight Orchid using plant vitrification solution 2 (PVS2). Measured parameters included the effect of PLB size (1-2 and 3-4mm), the effect of sucrose preculture concentrations and duration (0, 0.10, 0.25, 0.50 and 0.75 M, for 24 and 48 hours), the effect of dehydration duration (0, 10, 20, 30 and 40 minutes) and the effect of various unloading periods (0, 10, 20, 30 and 40 minutes) on survival of cryopreserved PLBs, as assessed using spectrophotometric 2,3,5-triphenyltetrazolium chloride (TTC) assay at 490nm. The PLBs were also subjected to a histological study to observe differences in both cryopreserved and non-cryopreserved PLBs of the orchid. The best results in the cryopreservation of PLBs of Vanda Kaseem's Delight was obtained when 3-4mm PLBs were precultured in VW medium supplemented with 0.1M sucrose for 24 hours, followed by a loading treatment, and 20 minutes of dehydration in PVS2 at 0°C, prior to cryostorage, and 30 minutes of unloading treatment after 90 seconds of thawing. Histological observations of cryopreserved PLBs indicated that most of the damages resulting from cryostorage occurred in the cell wall and nucleus of the cells

A leaf cross-section from <i>L</i>. <i>discolor</i> displaying cells with multiple anthocyanic layers.

<p>The arrangement of cells containing chloroplasts (C) and anthocyanins (A) were observed under brightfield microscopy (<b>a</b>), UV rays (<b>b</b>), blue light (<b>c</b>) and green light (<b>d</b>). AB = abaxial; AD = adaxial; CU = cuticle; PM = palisade mesophyll; S = stoma; SM = spongy mesophyll. Bar = 100 μm.</p