Jessica Jeyanthi James Antony

Preliminary study on cryopreservation of Dendrobium Bobby Messina protocorm- like bodies by vitrification

Although the delayed-type hypersensitivity (DTH) skin reaction to tuberculin is used worldwide for tuberculosis (TB) detection, it has poor diagnostic specificity due to the presence of common antigens in tuberculin shared by many mycobacterial species. The problem is noticed, especially in countries where the Bacillus Calmette-Gue´rin (BCG) vaccination is widely practiced. Thus, a new skin test antigen specific for the diagnosis of Mycobacterium tuberculosis (MTB) infection is urgently needed. CFP-10, a mycobacterial secretary protein that is absent in Mycobacterium bovis BCG and most other mycobacterial species including Mycobacterium avium, Mycobacterium intracellulare, has been shown to elicit cellular immune responses in MTB infected individuals and can be a good candidate for MTB specific diagnosis. We prepared recombinant MTB CFP-10, rCFP-10, and its utility as specific antigen for TB diagnosis was evaluated by skin testing in guinea pigs sensitized with M . tuberculosis, M. bovis, and M. bovis BCG. Our results show that the purified MTB rCFP-10 antigen elicits a positive skin response only in the guinea pigs sensitized with M. tuberculosis and M. bovis , and not in the animals sensitized with M. bovis BCG vaccine. The data presented in this study supports further testing of the use rCFP-10 as the specific antigen in the skin test for the diagnosis of MTB infection in humans. Key words : Recombinant CFP-10 protein, skin test, delayed-type hypersensitivity, tuberculosis infection, Mycobacterium tuberculosis, Mycobacterium bovis, Bacillus Calmette-Gue´rin.

Agrobacterium-Mediated Transformation of the Recalcitrant Vanda Kasem's Delight Orchid with Higher Efficiency

The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasems Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKDs PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A 600nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 𝜇M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

Effect of Plasmolysis on Protocorm-Like Bodies of Dendrobium Bobby Messina Orchid Following Cryopreservation with Encapsulation–Dehydration Method

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.

Characterization of the Second Generation Cryopreserved Dendrobium Bobby Messina Using Histological and RAPD Analyses

This study was conducted to detect the morphological, histological and molecular diff erences in the second generation of the PVS2 cryopreserved Dendrobium Bobby Messina [DBM] (18 months old culture) plantlets. Morphological analyses indicated that similarities and diff erences in cryopreserved DBM plantlets comparing to control stock culture based on selected morphological criteria. Morphological criteria, such as root length, number of shoot per explant and shoot length displayed diff erences, while the other three criteria, leaf diameter, leaf length and PLBs size were similar in cryopreserved compared to the control stock culture plant. Higher amount of homogenous cell population and denser cytoplasm were observed in cryopreserved PLBs compared to control stock culture PLBs based on histological analysis. This suggests the existance of somatic embryogenesis development mechanism taking place during the recovery and regeneration of the cryopreserved PLBs. However, RAPD analyses based on 10 primers indicated that cryopreserved DBM regenerated from vitrifi cation method generated a total of 20 to 39.9% polymorphic bands as compared to stock culture indicating potential somaclonal variation. Hence, an increase percentage of polymorphics bands in cryopreserved plantlets 18 months post cryopreservation as compared to previous report of 10% polymorphic bands in cryopreserved DBM 3 months post cryopreservation.