Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb.f.) christenson by encapsulation-dehydration

Abstract

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulationdehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth.

Cryopreservation of Cat’s Claw (<i>Uncaria tomentosa</i>)

“…Staining is fast and inexpensive and results can be used to predict the success of treatments [57] [58]. Staining with triphenyltetrazolium chloride (TTC), a colorless compound that is reduced to red-colored formazan by dehydrogenase enzymes in the mitochondria of living cells, is used mainly in seeds, embryos and meristems; however, the reliability of this test can be affected by other inorganic reactions that increase the production of formazan [58] [59]. The viability test using Evans blue allows differentiation of live and dead cells based on the selective permeability of the cellular membrane.…”

Conservación de plantas regeneradas in vitro y análisis de la variación somaclonal de Cinchona officinalis, Linneo

“…2008), en Rabdosia rubescens, se recuperaron el 85% de meristemas crioconservados (Ai et al 2012) y en Phalaenpsis bellina se alcanzó un máximo de sobrevivencia de 47% con un 30 % de rebrote luego de la descongelación utilizando la encapsulación-deshidratación como método crioprotector (Khoddamzadeh et al 2011). En variedades de pistacho tuvieron en…”

“…2) Crioconservación de tejidos cultivados in vitro, es la paralización del crecimiento por periodos indefinidos Khoddamzadeh et al 2011;Ai et. 2011;Bekheet 2011;Cruz et al 2013).…”

Cryopreservation of Orchid Genetic Resources by Desiccation: A Case Study of Bletilla formosana

“…Prior to ultralow temperature preservation, suitable pretreatment methods are used to increase the survival rate of the materials to be preserved. Three pretreatments, namely desiccation, vitrification, and encapsulation-dehydration, are typically applied for orchids [13,[21][22][23][24]. Pretreatment technologies prior to cryopreservation are still a fancy work to investigate now.…”

“…Vitrification and desiccation methods have been often used to preserve seeds by removing water from the cells [9][10][11] because the water content of plant materials may affect cryopreservation success. Orchid PLB (protocorm like body) conservation by combining encapsulation and dehydration has been suggested [12][13][14].…”