Effect of Cryopreservation Method Supported With Biochemical Analyses in the Axillary Bud of Jewel Orchid, Ludisia Discolor

This study was conducted to investigate the potential of conserving an endangered terrestrial jewel orchid Ludisia discolor using in vitro grown axillary buds. Excised segments of axillary buds (4 - 5 mm in length) were precultured on a modified Murashige and Skoog (MS) medium supplemented with 0.2 M sucrose for 24 h and osmoprotected in a loading solution for 20 min. Then, axillary buds were dehydrated in PVS2 solution for 10 min at 0°C and incubated in liquid nitrogen for 1 h. Subsequently, axillary buds were rewarmed rapidly by dilution solution and transferred to a growth recovery medium supplemented with 0.05 µM melatonin under in vitro conditions that led to an improved survival chance (16.67%) for cryopreserved L. discolor. The abiotic stresses and the overproduction of reactive oxygen species (ROS) during cryopreservation stages may contribute to cryoinjuries and poor survival. The varied response towards stress was detected with significantly increased values recorded at certain cryopreservation stages, including proline activity at the dehydration stage (5.51 µmol/g), catalase at the preculture (85.64 U/g) and dehydration (70.87 U/g) stages, peroxidase at the rewarming stage (565.37 U/g) and ascorbate peroxidase during the loading stage (12.19 U/g). Hence, this first attempt to cryopreserved L. discolor indicates that future experimental designs could include exogenous antioxidants and different vitrification solutions to improve survival and regeneration.

Comparison of matK and rbcL DNA barcodes for genetic classification of jewel orchid accessions in Vietnam

Background Jewel orchid is the common name of several orchid species which can be alike in morphological characteristics, but variable in medicinal properties. At present, two DNA barcode loci, namely, maturase K (matK) and ribulose 1,5-biphosphate carboxylase (rbcL), are intensively utilized for plant identification. However, the discrimination effectiveness of these loci is variable among plant species. This study was carried out to compare the identifying efficacy of these two loci on jewel orchid population collected throughout Vietnam. Results The results revealed that 21 jewel orchid accessions studied were segregated into four different species with significant variations. The discrimination power of matK and rbcL markers in this jewel orchid study displayed different efficiency level. The rbcL gene has higher distinguishing potential than either matK gene alone or the combination of both genes. Conclusion The findings of this project could provide valuable information that is necessary for classification, plant origin identification, breeding, and conservation program of jewel orchid in Vietnam.

Corybas boholensis (Orchidaceae): A New Jewel Orchid Species from Bohol Island, Central Visayas, Philippines

A new, critically endangered jewel orchid species, Corybas boholensis, is described and illustrated from forest over limestone on Bohol Island, Philippines. This diminutive species, belonging to the C. pictus complex, is distinct in having a deep red flower and a truncate, warty dorsal sepal with 9–11 veins at its widest point near the apex. This is the fifth species of Corybas recorded for the Philippines.

Effect of colchicine and oryzalin treatment on polyploid induction and morphogenesis of Anoectochilus setaceus Blume cultured in vitro

Jewel orchid (Anoectochilus setaceus Blume) is one of the important medicinal plants used for many common diseases treatment such as hypertension, diabetes, and heart, liver and lung diseases, as well as improving the health in general. Polyploid induction using colchicine and oryzalin has been widely performed in various types of crops and could be considered a valuable tool for plant breeding. In the present study, in vitro young shoots of A. setaceus Blume (1 cm in height) were treated with different concentrations of colchicine (0, 1, 2, 4, 6, 8, and 10 µM) and oryzalin (0, 10, 20, 40, 60, 80, and 100 µM) at different durations (0, 12, 24, 36, and 48 h). The results showed that shoots with length of treatment from 24 to 48 h and the concentrations from 2 to 10 µM of colchicine and from 40 to 100 µM of oryzalin appeared morphological differences (10.00% - 16.00%) but the tendency to return to normal of these shoots after several times of sub-culture was observed. Stomatal length had a close correlation with the level of ploidy. Diploid plantlets (2n = 2x) had the average stomatal length of 33.50 µm when observed under an optical microscope at 100x magnification and corresponding to the peak at 70 FL in flow cytometric DNA histogram. In all identified polyploid plantlets, the stomatal lengths were greater than 40.12 µm. Moreover, flow cytometric analysis indicated that plantlets with stomatal lengths ranged from 40.12 to 50.00 µm were diploid-tetraploid mosaicisms represented by two separate peaks at 70 FL and 90 FL. Finally, samples treated with 4, 6, and 8 µM colchicine with treatment time of 48 h, and 60 µM oryzalin with treatment time of 24 h that had stomatal lengths ranged from 52.03 to 71.25 µm were determined to be tetraploid (2n = 4x) represented by a peak at 120 FL position.