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Sopalun K.,Mahidol University | Thammasiri K.,Mahidol University | Ishikawa K.,Japan Horticultural Production and Research Institute
Cryo-Letters | Year: 2010

Three vitrification-based methods for the cryopreservation of Grammatophyllum speciosum protocorms were invesigated: droplet-vitrification, encapsulation-dehydration and encapsulation-vitrification. Protocorms, 0.1 cm in diameter, developed from 2-month-old germinating seeds were used. For droplet-vitrification, protocorms were precultured on filter paper soaked in half strength Murashige and Skoog medium (1/2MS) containing 0.4 M sucrose at 25 ± 2°C for 2 d, followed by soaking in loading solution (2 M glycerol and 0.4 M sucrose in 1/2MS liquid medium) for 20 min and then dehydrated with PVS2 solution [30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethyl sulfoxide in 1/2MS liquid medium containing 0.4 M sucrose at pH 5.7] for 30 min. For encapsulation-dehydration, encapsulated protocorms were precultured in 1/2MS liquid medium containing 0.4 M sucrose on a shaker (110 rpm) at 25 ± 2°C for 2 d, followed by soaking in the same loading solution for 20 min and then exposed to a sterile air-flow at 2.5 inches/water column from the laminar air-flow cabinet for 8 h. For encapsulation- vitrification, encapsulated protocorms were precultured in 1/2MS liquid medium containing 0.4 M sucrose for 1 or 2 d, followed by soaking in the same loading solution for 20 min and then dehydrated with PVS2 solution for 60 min. For all three methods, preculturing with 0.4 M sucrose for 2 d resulted in a significant induction of dehydration and freezing tolerance. The cryopreservation results showed highest protocorm regrowth after droplet-vitrification (38%), followed by encapsulation-dehydration (24%) and encapsulation-vitrification (14%). Plantlets developed from these three methods did not show any abnormal characteristics or ploidy level change when investigated by flow cytometry. © CryoLetters. Source


Jitsopakul N.,Mahidol University | Thammasiri K.,Mahidol University | Ishikawa K.,Japan Horticultural Production and Research Institute
Acta Horticulturae | Year: 2011

Vanda coerulea is the most popular Vanda species of Thailand that has become endangered because of the wild orchid trade, deforestation and environmental changes. To preserve this orchid, droplet-vitrification was studied for the cryopreservation of protocorm-like bodies. Protocorm-like bodies were developed from one protocrom in ND liquid medium supplemented with 1 mg/L BA in combination with 0.5 mg/L NAA and 30 g/L maltose. Protocorm-like bodies (3.0 mm in diameter) were precultured in ND liquid medium supplemented with 0.5 M sucrose for 1 day on a shaker, then dehydrated with loading solution for 15 min and exposed to PVS2 solution for 30 min at 25°C. The plant materials were then immersed in liquid nitrogen, rewarmed rapidly and cultured on solidified ND medium. After cryopreservation, the survival frequency of cryopreserved protocorm-like bodies was 5%. The assessment of the genetic stability of cryopreserved protocorm-like bodies was developed using RAPD markers, size and sequence of the trnL (UAA) non-coding region of cpDNA. Results showed the same RAPD patterns of non-cryopreserved and cryopreserved protocorm-like bodies. The size and sequences of the trnL (UAA) non-coding region of cpDNA for noncryopreserved and cryopreserved protocorm-like bodies were not different. There was no difference in morphology and similar patterns of ploidy in plantlets developed from non-cryopreserved and cryopreserved protocorm-like bodies. Source


Sopalun K.,Mahidol University | Thammasiri K.,Mahidol University | Ishikawa K.,Japan Horticultural Production and Research Institute
World Academy of Science, Engineering and Technology | Year: 2010

The effects of chitosan, a biodegradable polymer, were studied in Grammatophyllum speciosum protocorm-like bodies (PLBs) in vitro culture. The chitosan concentration of 0, 5, 10, 15, 20, 25, 50 or 100 mg/l were supplemented in half-strength Murashige and Skoog (1/2 MS) liquid or on agar media containing 2% (w/v) sucrose. The results showed that liquid medium supplemented with 15 mg/l chitosan showed the highest relative growth rate (7-fold increase) of PLBs. On 1/2 MS agar medium supplemented with 25 mg/l chitosan gave the highest relative growth rate (4-fold increase). The relative growth rate of G. speciosum PLBs on agar medium was significantly lower than that in liquid medium. Moreover, chitosan, supplemented to agar medium promoted shoot formation but not rooting. However, supplementation at too high a level, such as 100 mg/l can inhibit growth and kill PLBs. Source


Shishido M.,Chiba University | Sato K.,Japan Horticultural Production and Research Institute | Yoshida N.,Chiba University | Tsukui R.,Chiba University | Usami T.,Chiba University
Journal of General Plant Pathology | Year: 2010

We developed polymerase chain reaction (PCR) assays to detect and quantify Phomopsis sclerotioides, the causal agent of black root rot of cucurbits. We used internal transcribed spacers 1 and 2 of the ribosomal DNA (rDNA) from representative isolates to search for target sequences. Primer pairs were selected after testing against 40 fungal isolates including 13 Ph. sclerotioides isolates, 9 Phomopsis isolates other than Ph. sclerotioides, and 18 soilborne fungi that were either pathogenic or nonpathogenic to cucurbits. Conventional PCR assays with the primer pair of CPs-1 (forward) and CPs-2 (reverse) produced target DNA amplicons from all Ph. sclerotioides isolates but none of the other isolates tested. From soil and root samples collected from fields naturally infested with black root rot of cucumber and melon, the CPs-1/CPs-2 primer pair successfully amplified target DNA fragments in conventional PCR assays. Moreover, we applied the CPs-1/CPs-2 primer pair in a real-time PCR assay with SYBR Green I, and PCR-amplified products were successfully quantified by reference to a standard curve generated by adding known amounts of target DNA. Target Ph. sclerotioides DNA fragments were similarly detected in artificially inoculated roots of cucumber, melon, pumpkin, and watermelon, but quantities of Ph. sclerotioides DNA in their hypocotyls of the hosts varied as follows: melon ≥ cucumber ≥ watermelon > pumpkin. These results suggest that Ph. sclerotioides infection is not species-specific but the rate of infection may differ among host species. © 2009 The Phytopathological Society of Japan and Springer. Source


Sopalun K.,Mahidol University | Thammasiri K.,Mahidol University | Ishikawa K.,Japan Horticultural Production and Research Institute
Plant Cell, Tissue and Organ Culture | Year: 2010

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry. © 2010 Springer Science+Business Media B.V. Source

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