This study investigated 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 plant vitrification solution 2 (PVS2) 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, which led to an improved survival chance (16.67%) for cryopreserved L. discolor. The osmotic stress and the overproduction of reactive oxygen species (ROS) during cryopreservation stages may result in cryoinjuries and poor survival as increased levels of proline (5.51 µmol/g), catalase (85.64 U/g), peroxidase (565.37 U/g), and ascorbate peroxidase activities (12.19 U/g) were detected after dehydration, preculture, rewarming, and loading stage, respectively. Results obtained from this study indicate that further experimental designs which apply different PVS and exogenous antioxidants are needed for improved survival and regrowth of L. 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.

Ludisia discolor, a jewel orchid is the only species found under its genus (Ludisia). This wild orchid is well known for its striking foliage and its medicinal benefits. Since the population of this valuable species is becoming scarce, therefore protecting this orchid is crucial. Cryopreservation is a practicable long-term germplasm conservation approach. The genetic material is stored at freezing temperature using liquid nitrogen. In this study, V cryo-plate method was developed using L. discolor axillary buds using optimum conditions. For the screening of somaclonal variation, 3 weeks old treated (cryopreserved and non-cryopreserved) explants were utilized in comparison with the stock cultures as a control. The genetic stability was assessed using a total of twenty (20) DAMD, twenty (20) ISSR, and ten (10) SCoT molecular markers. The somaclonal variation detected in cryopreserved explant using DAMD, ISSR, and SCoT molecular markers were 12.5, 4.17, and 10.53% respectively. However, somaclonal variation was also detected in non-cryopreserved explants using DAMD, ISSR, and SCoT at 6.15, 5.77, and 10%, respectively. Hence, SCoT was chosen to be precise than the other two molecular markers (DAMD and ISSR).