A preliminary study on the germination of Eurycoma Longifolia Jack (Tongkat Ali) seeds

Eurycoma longifolia fruits are borne in large bunches consisting of 200-300 fruits in each bunch. Even though trees produce abundant fruits during the peak fruiting season in September, yet the number of seedlings found growing in the forest floor is very low. Our results indicated that the seeds with hard endocarp sown in a 1:1 soil and sand mixture only start to germinate 43 days after sowing and continue to germinate over a period of 99 days. The soil and sand mixture (1:1) is equivalent to that of the forest sandy soil combination that was optimum for germination of Eurycoma longifolia seeds. The seeds with endocarp intact that are sown in jiffy germinated within 35-85 days. However, when the endocarp of seeds were removed, the seeds germinated within two weeks via in vitro culture using basic MS medium. The npe seeds germinated better when sown in the 1:1 soil and sand mixture than in jiffy pellets. But the unnpe seeds with the endocarp removed seemed to germinate faster (cve = 0.2053) when cultured in vitro in basic MS medium. All the seedlings were found to have the same growth pattern in terms of seedling height, number of leaves produced, and the stem diameter irrespective of germination methods over a period of 120 days

Effects of Medium Constituents on Growth and Canthinone Accumulation in Cell Suspension Cultures of Eurycoma longifolia Jack

The effect of various macronutrients, micronutrients and sucrose on growth and canthinone alkaloid production in cell suspension cultures of Pasak Bumi (Eurycoma longifolia Jack) was investigated. The optimum macronutrients and micronutrients content for the high alkaloid production of E. longifolia Jack was different to that found in the Murashige and Skoog (MS) medium. The highest amount of alkaloids, 9-hydroxycanthin-6-one and 9-methoxycanthin-6-one, could be obtained from E. longifolia Jack cells cultured in modified MS liquid medium that containing macronutrients: 21.50 mM NH4NO3, 14.25 mM KNO3, 7.50 mM CaCl2•2H2O, 2.50 mM MgSO4•7H2O, 1.45 mM KH2PO4, while content of micronutrients was 0.233 mM FeNa-EDTA, 0.215 mM MnSO4•4H2O and without CuSO4•5H2O. Increased sucrose concentration to 4.00% (w/v) in modified MS liquid medium could increase total of two-alkaloid. The modification of macronutrients and micronutrients concentration based the optimum production of biomass was obtained MSBs medium that producing high biomass but also increasing the production of 9-hydroxycanthin-6-one. The modification of macronutrients or macronutrients and micronutrients based the optimum total of two-alkaloid was obtained MSC and MSD medium that producing low fresh weight but producing the high 9-hydroxycanthin-6-one. Key words: Pasak Bumi, 9-hydroxycanthin-6-one, 9-methoxycanthin-6-one, macronutrients, micronutrients, sucros

Effects of ascorbic acid on PVS2 cryopreservation of dendrobium Bobby Messina’s PLBs supported with SEM analysis.

Regrowth of the cryopreserved protocorm-like bodies (PLBs) of Dendrobium Bobby Messina was assessed based on the plant vitrification solution 2 (PVS2) optimisation conditions. The optimized protocol obtained based on TTC spectrophotometrical analysis and growth recovery were 3–4 mm of PLBs size precultured in 0.2 M sucrose for 1 day, treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min and subsequently dehydrated with PVS2 at 0 °C for 20 min prior to storage in liquid nitrogen. Following rapid warming in a water bath at 40 °C for 90 s, PLBs were treated with unloading solution containing half-strength liquid MS media supplemented with 1.2 M sucrose. Subsequently, the PLBs were cultured on half-strength semi-solid MS media supplemented with 2 % (w/v) sucrose without any growth regulators and resulted in 40 % growth recovery. In addition, ascorbic acid treatment was used to evaluate the regeneration process of cryopreserved PLBs. However, growth recovery rates of non-cryopreserved and cryopreserved PLBs were 30 and 10 % when 0.6 mM ascorbic acid was added. Scanning electron microscopy analysis indicates that there are not much damages observed on both cryopreserved and non-cryopreserved PLBs in comparison to PLBs stock culture

Effect of Basal Medium on In Vitro Leaf Morphology, Growth and Artemisinin Production of Artemisia annua L.

Artemisia annua L. was classified as one of the important medicinal plants due to its potential in the treatment of malaria. However, the propagation of this plant was limited by environmental and geographical factors. Therefore, in vitro culture technique was an alternative to overcome these limitations. Five different basal media were examined for their effect on the growth and artemisinin content of in vitro plantlets of A. annua. They were found to give different effect on the growth in term of height, fresh biomass and rooting ability of the plantlets. Glandular and non-glandular filamentous trichomes were observed on the adaxial and abaxial surface of A. annua leaf. The five basal media was found to affect the distribution and the number of trichomes and stomata formed on the leaf surfaces. LV medium induced more trichomes formation of both types on both leaf surfaces. Highest number of stomata was found on the leaf surface of the plantlets cultured in MS medium. While B5 medium resulted in non formation of stomata on the abaxial leaf surface of all the studied clones. Artemisinin production was found to greatly affect by the choice of basal medium used for cultivation. Keywords: Artemisia annua, artemisinin, basal medium, growth pattern, stomata, trichome

Preliminary study of DMSO vitrification technique of Dendrobium sonia 28 using protocorm-like bodies (PLBs) explant

In vitro grown shoot derived from protocorm-like bodies of Dendrobium sonia 28 hybrid were cryopreserved under liquid nitrogen condition, by means of DMSO vitrification method. Prior to the cryopreservation, the shoot were excised into two types of different length of 0.5-1.0 cm and 1.0-1.5 cm. Those entire excised shoot were grown on half-strength Murashige and Skoog (MS) semi solid medium. Upon DMSO vitrification method, the shoots were precultured (24 and 48 hours) at different sucrose concentrations (0.06 M, 0.10 M, 0.25 M, 0.50 M and 0.75 M). Vitrification process proceeded with culturing of the shoots in a loading solution consist of mixture of 2 M glycerol and 0.4 M sucrose for 20 minutes at room temperature (28°C), followed by further dehydration process with DMSO for a different incubation duration (0 minute, 10 minutes, 20 minutes and 30 minutes) at temperature of 0°C and 24°C. The shoots were later plunged into liquid nitrogen. After recovering from the liquid nitrogen storage, the shoots underwent rapid thawing (40°C) and were grown in a regrowth semi solid medium for two days under dark condition. TTC analysis was carried out to determine the viability of the shoots after storage under liquid nitrogen. The highest absorbance value at 540 nm was obtained using 2,3,5, triphenyl tetrazolium chloride (TTC) assay from the treatment of 1.0-1.5 cm shoots precultured for 24 hours in 0.5 M sucrose concentration MS semi-solid medium at 0°C for the 10 minutes incubation time in DMSO solution. The DMSO vitrification method was a crucial step in the orchid cryopreservation of Dendrobium sonia 28 hybrid shoot. Treatment of DMSO solution was proven to be capable of carrying out the dehydration process which was important for the survival rate of Dendrobium sonia 28 hybrid shoot undergoing cryopreservation at ultra low temperature (-196°C)

Preliminary study on cryopreservation of Dendrobium Bobby Messina protocorm- like bodies by vitrification

Protocorm-like bodies of Dendrobium Bobby Messina were cryopreserved by vitrification method. In this study, protocorm-like bodies (PLBs) with the size range of 1-2 and 3-4 mm were selected from 4 weeks old culture, pretreated with half strength semi-solid Murashige and Skoog (MS) media supplemented with 0.5 M sucrose at 25°C for 24 h. Pretreated PLBs were then treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half strength liquid MS media at 25°C for 20 min. Osmoprotected PLBs were then dehydrated with plant vitrification solution 2 at 0°C for 20 min before storage in liquid nitrogen. After rapid warming in water bath at 40°C for 90 s, the PLBs were washed with half strength liquid MS media supplemented with 1.2 M sucrose and then cultured on half strength semi-solid MS media supplemented with 2% sucrose without the presence of any growth regulators. Survival of the cryopreserved PLBs was assessed based on triphenyl tetrazoliumchloride (TTC) spectrophotometrical analysis. The PLBs with 3-4 mm size range showed better viability comparative to size range 1-2 mm for both cryopreserved and non-cryopreserved PLBs. The best pretreatment concentration used in pretreatment media was 0.6 M sucrose and 1.2 M sorbitol, respectively

Preliminary study on cryopreservation of Dendrobium Bobby Messina protocorm- like bodies by vitrification

Protocorm-like bodies of Dendrobium Bobby Messina were cryopreserved by vitrification method. In this study, protocorm-like bodies (PLBs) with the size range of 1-2 and 3-4 mm were selected from 4 weeks old culture, pretreated with half strength semi-solid Murashige and Skoog (MS) media supplemented with 0.5 M sucrose at 25°C for 24 h. Pretreated PLBs were then treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half strength liquid MS media at 25°C for 20 min. Osmoprotected PLBs were then dehydrated with plant vitrification solution 2 at 0°C for 20 min before storage in liquid nitrogen. After rapid warming in water bath at 40°C for 90 s, the PLBs were washed with half strength liquid MS media  supplemented with 1.2 M sucrose and then cultured on half strength semi-solid MS media supplemented with 2% sucrose without the presence of any growth regulators. Survival of the cryopreserved PLBs was assessed based on triphenyl tetrazoliumchloride (TTC) spectrophotometrical analysis. The PLBs with 3-4 mm size range showed better viability comparative to size range 1-2 mm for both cryopreserved and non-cryopreserved PLBs. The best pretreatment concentration used in pretreatment media was 0.6 M sucrose and 1.2 M sorbitol, respectively.Key words: Cryopreservation, vitrification, Dendrobium Bobby Messina, Pretreatment

Chemotaxis movement assay of Eurycoma longifolia using wild and disarmed strains of Agrobacterium rhizogenes

Bacterial chemotaxis is considered the first step in the interaction between motile bacteria and plant cells. Chemotaxis initiates the process of bacterial infection towards the plant cells and thus conferring beneficial attributes to the host. In this study, 5 wild strains and 2 disarmed strains of Agrobacterium rhizogenes were tested for chemotaxis assay using the swarm agar plate method. As expected, strong positive chemotactic response was observed in most of the tested bacteria strains and all the tested strains of Agrobacterium rhizogenes showed positive chemotactic response towards the tested root and somatic embryos of the valuable medicinal plant, Eurycoma longifolia. Therefore, induction of hairy roots is possible in Eurycoma longifolia. Generating hairy roots in Eurycoma longifolia will be highly beneficial mainly to the pharmaceutical industry as this medicinal plant possesses the capacity to produce many secondary metabolites which is proposed to increase sexual virility properties and to have anti cancer properties