In vitro multiplication of the rare and endangered slipper orchid, Paphiopedilum rothschildianum (Orchidaceae)

Paphiopedilum rothschildianum is an endangered orchid species endemic to Mount Kinabalu, Sabah, and Malaysia. The vegetative propagation of this plant has always been restricted due to its slow growth and maturation rates. Thus, an in vitro tissue culture technique was explored in order to overcome this limitation. In this study, clonal propagation of P. rothschildianum was achieved through in vitro formation of multiple shoots from stem nodal and single shoot explants cultured onto halfstrength Murashige and Skoog medium. The responses of the explants to the presence of different types of organic nitrogen additives viz. casein hydrolysate, peptone and tryptone-peptone (in amount of 0.5, 1.0 and 2.0 g/l) in the culture medium were also evaluated. The addition of these organic nitrogen additives into the basal medium slightly enhanced the number of multiple shoots formed on both types of explants when compared to additive-free MS medium. After 16 weeks of culture, an average of 2.9 shoots per stem nodal explant and 2.8 shoots per single shoot explant were obtained on half-strength MS medium supplemented with 1.0 g/l peptone and 2.0 g/l tryptone-peptone, respectively. All the newly-formed shoots were divided into single plantlets and subcultured onto similar respective medium. After an additional 12 weeks of culture on the same medium, plantlets with 3 - 4 roots were acclimatized and transferred to a glass house where they showed 90% survival rate. Thus, the method presented in this study had provided a promising strategy for the production of large numbers of phenotypically stable P. rothschildianum

Responses of some selected Malaysian rice genotypes to callus induction under in vitro salt stress

Tissue culture technique can be used as a source for genetic variability by means of genetic modifications through the process of in vitro cultures. This technique has been widely used for breeding purposes, especially for stress tolerance selection, which severely limits rice production. Also, the establishment of a suitable plant regeneration system is a prerequisite for successful genetic transformation. The aim of this study is to identify the most suitable medium and to assess the genotype performance for in vitro salt stress responses in some selected Malaysian rice genotypes. Differences in culture conditions, growth rate, plant hormone responses and accumulation of proline content were monitored. All the selected genotypes showed that the callus-growth capacities were significantly affected by the genotypes and the culture media. Evidently, callus was best induced on the MS medium added with 10 ìM dichlorophenoxyacetic acid (2,4-D) and 0.4 gm/l casein hydrolysate. In addition, the shoot regeneration capacity from the callus was the most effective in ½ MS added with 10 ìM 6 benzylaminopurine (BAP). The two genotypes, that is, MR219 (line 4) and MR219 (line 9), consistently performed the best in both callus culture (93.51 and 92.22%) and plant regeneration capacity (27.03 and 26.34%), respectively. When the callus were transferred to different concentrations of NaCl (0 to 250 mM) supplemented medium in order to examine their responses to salinity, the two genotypes, that is, MR219 (line 4) and MR219 (line 9), showed a significant decline in the callus growth (18.83 and 23.5%) and regeneration capacity (7.33 and 7.68%), respectively. A similar trend was also observed for the proline content. All the genotypes significantly resulted in proline accumulation.MR211 showed the highest accumulation, whereas MR219 (line 4) revealed the lowest proline accumulation. These proline content analyses further suggest potential salinity tolerance in the rice genotypes.Key words: Plant regeneration, embryogenesis, salinity, callus

RAPD analysis of colchicine induced variation of the Dendrobium Serdang beauty

Variation was detected in Dendrobium Serdang Beauty V (DSB V) plantlets regenerated from protocorm like bodies (plbs) induced by various concentration levels of colchicines in the Murashige and Skoog media (MS) supplemented with 1.5 mg/L IBA. RAPD analysis detected 6 - 26% variation in the regenerants from the mother plant. The highest variation was obtained in regenerates treated with 25 mg/L colchicine, which also exhibited reduced regeneration rates from plbs and mean plantlet fresh weight. RAPD analysis also showed high polymorphism between the mutated regenerant DSB V, and 13 species of the Dendrobium genera, and 13 orchids across generas. However, despite the 26% colchicine induced variation in the regenerants, all RAPD analysis revealed that DSB V was closely related to the mother plant. Thus, the RAPD technique is favourable for variation detection as it was sensitive enough to detect variations at species level and among somaclonal variants in this study

Establishment of a plant regeneration system from callus of Dendrobium cv. Serdang Beauty

An in vitro propagation protocol was established for the Dendrobium Serdang Beauty orchid. The propagation protocol utilized calli tissues that were successfully initiated from protocorm-like bodies(PLBs) explants, while the leaf and root tip explants died. The percentage of protocorm-like bodies explants responding to calli formation was 100% in all tested levels of IAA, IBA and NAA auxintreatments. The highest amount of calli (49.59 gram) proliferated on MS medium containing 1.5 mg/L IBA. These calli successfully regenerated on media supplemented with either KIN or BAP cytokinins and combined treatments of KIN and IAA (4 mg/L) or NAA (1.5 mg/L). However, media supplemented with only 1 mg/L KIN was sufficient to produce significantly high percentage of plantlet formation (80%),high number of planlets per explant (4-5 plantlets) and high mean fresh weight per plantlet (11.128 g). These plantlets were acclimatized on all tested media and obtained satisfactory rate of plantlet survival(80-100%), mean number of leaves per plant (4-6 leaves), and mean leaf length (4 - 5 cm). Among these media, charcoal was considered the most economical and available material in the local market. Duringthe development of this protocol, substantial necrosis of calli were observed when cultures were treated with 2,4-D and BAP. It was proposed that the presence of ethylene within the cultures, which isknown to be emitted by plant growth regulators into the micro-climate of in vitro culture vessels, is the determining factor of a suitable plant growth regulator for the survival and growth of the DendrobiumSerdang Beauty calli cultures in our study