Preliminary investigation of cryopreservation by encapsulation-dehydration technique on

Protocorm-like bodies (PLBs) of Brassidium Shooting Star, a new commercial ornamental orchid hybrid, were cryopreserved by an encapsulation-dehydration technique. The effects of PLB size, various sucrose concentrations in preculture media and sodium alginate concentration for encapsulation were the main parameters assessed. Four-week old PLBs (1 to 2 and 3 to 4 mm) were precultured in half strength semi-solid Murashige and Skoog (MS) media supplemented with six different sucrose concentrations (0, 0.2, 0.4, 0.6, 0.8 and 1.0 M) for 24 h, followed by encapsulation in 2.5, 3.0 or 3.5% sodium alginate, with 0.1 M calcium chloride been used as the hardening agent. The beads formed were then osmoprotected in half-strength liquid MS media supplemented with 0.75 M sucrose and dehydrated for three hours in 50 g heat-sterilized silica gel before cryostorage in sterile cryovials. The beads were thawed in a 40 ± 2°C water bath and then directly placed in recovery media for two weeks under tissue culture conditions. After two weeks of recovery, the survival rates of the encapsulated PLBs were evaluated by the 2,3,5-triphenyltetrazolium chloride (TTC) assay. The best conditions for the encapsulation-dehydration of Brassidium Shooting Star were discovered to be the preculture of 3 to 4mm PLB in half strength semi-solid MS media supplemented with 0.8 M sucrose, followed by encapsulation in 3.5% sodium alginate. Further biochemical analysis (chlorophyll, total soluble protein and peroxidases activities) were conducted to investigate the physiological responses of the PLBs after cryopreservation.Key words: Encapsulation-dehydration, cryopreservation, Brassidium Shooting Star, protocorm-like bodies

Preliminary investigations of Agrobacterium-mediated transformation in indica rice MR219 embryogenic callus using gusA gene

Preliminary steps in the genetic transformation of indica rice MR219 was investigated in the plant- Agrobacterium tumefaciens interaction. Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector pCAMBIA 1305.2 harboring the modified GUS gene driven by the CaMV 35S promoter was used. Various transformation parameters influences were optimized using embryogenic calli via β- glucuronidase (GUS) as a reporter marker. Various transformation parameters were optimized including bacterial concentration, age of embryogenic callus, pre-culture period, wounding technique, cocultivation period, immersion time and dry time before co-cultivation, acetosyringone (AS) concentration, pH of co-cultivation media and temperature of the co-cultivation period. The expression of the transient gusA gene in the plant genome was preliminary confirmed by histochemical GUS assay activity (as blue spots). The results from transient gusA gene expression of calli suggested that the Agrobacterium-mediated transfer system of T-DNA in indica rice MR219 was highly efficient. Therefore, the investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of indica rice MR219 calli.Key words: Indica rice MR219, Agrobacterium tumefaciens, GUS expression

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