Protocorm-like bodies (PLBs) of Brassidium Shooting Star orchid were successfully cryopreserved using droplet-vitrification method. Vitrification based cryopreservation protocol is comprised of preculture, osmoprotection, cryoprotection, cooling, rewarming, and growth recovery and each and every step contributes to the achievement of successful cryopreservation. In order to reveal the lethal and nonlethal damage produced by cryopreservation, histological observation, scanning electron microscopy (SEM), and biochemical analysis were carried out in both cryopreserved and noncryopreserved PLBs of Brassidium Shooting Star orchid comparing with the control PLBs stock culture. Histological and scanning electron microscopy analyses displayed structural changes in cryopreserved PLBs due to the impact of cryoinjury during exposure to liquid nitrogen. Total soluble protein significantly increased throughout the dehydration process and the highest value was achieved when PLBs were stored in liquid nitrogen. Ascorbate peroxidase (APX) and catalase (CAT) showed the highest enzyme activities in both dehydration and cryostorage treatments indicating that stress level of PLBs was high during these stages.
Protocorm-like bodies (PLBs) of Dendrobium orchids are emerging as a potential source of valuable secondary metabolites. This study examined the effect of four additives namely 1-naphthaleneacetic acid (NAA), kinetin, thidiazuron (TDZ), and activated charcoal (AC) used in culture medium on genetic variability in PLBs of Dendrobium Sabin Blue. Nine ( 9) ISSR primers and eleven (11) DAMD primers were used to assess the genetic variability of PLBs that were subcultured over a period of two years. We confirmed that the use of kinetin in culture medium for two years resulted in the highest rate of somaclonal variation in PLBs. On the other hand, TDZ and activated charcoal registered the lowest genetic variability in PLBs. The findings of this study suggest the importance of selecting additives used in the culture medium to maintain stable genetic lines of PLBs. We recommend that the assessment of somaclonal variations should be performed for long term maintenance of tissue cultures.
Cryopreservation is an alternative, safe, and cost-effective method for long-term plant genetic resource conservation. This study was conducted to optimize the conditions for cryopreserving the protocorm-like bodies (PLBs) of Brassidium Shooting Star orchid with the PVS3 vitrification method. Five parameters were assessed in this study: PLB size, sucrose concentration, preculture duration, PVS3 duration, and unloading duration. The viability of the cryopreserved PLBs was determined using the triphenytetrazolium chloride assay and growth recovery assessments. The optimum condition for the cryopreservation of the PLBs of Brassidium Shooting Star orchid is based on the size range between 3 and 4 mm precultured with half-strength semi-solid MS media supplemented with 0.25 M sucrose for 24 h, followed by treatment with loading solution mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min. The PLBs were then dehydrated with PVS3 at 0 °C for 20 min prior to immersion in liquid nitrogen; finally, the PLBs were immersed with half-strength liquid MS media supplemented with 1.2 M sucrose for 30 min. Histological analyses displayed denser cytoplasm and voluminous nucleus in the cryopreserved PLBs of Brassidium Shooting Star orchid.
Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates the cellular process of cryopreserved protocorm-like bodies (PLBs) was different comparative to non-cryopreserved PLBs. The cellular process was not only modified by the freezing and thawing effect but also due to the dehydration process itself during the cryopreservation procedure. Histological observation in Dendrobium Bobby Messina in encapsulation-dehydration method indicated that the degree of plasmolysis causes more cellular changes to the cryopreserved PLBs comparative to non-cryopreserved and stock culture PLBs. These results revealed higher amount of homogenous cell population and denser cytoplasm in cryopreserved PLBs. Histological analysis also revealed more voluminous nucleus in cryopreserved PLBs comparative to non-cryopreserved PLBs and PLBs stock culture. In contrast, scanning electron microscope analysis showed severe damages in cryopreserved PLBs and non-cryopreserved PLBs comparative to the PLBs stock culture which in return could be the possible reason of no regrowth in encapsulation-dehydration method. Damages incurred were on top part, side part, and at the stomata of the PLBs. Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates that the degree of plasmolysis causes changes in the cellular process of PLBs from cryopreserved PLBs was different comparative to non-cryopreserved PLBs.
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