Retno Mastuti

The role of AUX1 gene and auxin content to the branching phenotype of Kenaf (Hibiscus cannabinus L.).

The objectives of this research were to identify auxin gene, AUX1, and to determine the plant auxin content and their role in conferring branching on Kenaf. PCR analysis using AUX1 primer capable to amplify the DNA of non branching (KR11) and branching kenaf mutant, resulting in 800 bp PCR product. The sequence of the PCR product showed high degree of homology with the sequence of AUX1 gene of other plants in the NCBI GenBank database, confirming kenaf possession of the gene AUX1. However, some variation on the DNA sequence was found between branching and non branching phenotype indicated allele differences of the same gene which were responsible for the variation in the type of branching. Identification of auxin content in the roots, apical shoot, and axillary branches using spectrophotometry method showed that the branching plant has higher auxin content in the apical shoot compared to the content in the branches. This indicate that AUX1 controls the formation of branches by controlling either the content of auxin in the apical shoot and branches, or the ratio of auxin content in the shoot and branches.

In vitro enzymatic isolation of protoplasts from tissues of the medicinal plant Physalis angulata L.

Physalis angulata is a medicinal plant from the Solanaceae family. Utilization of tissue culture, especially protoplast techniques, has potential for supplying medicinal plants in the framework of their development as standardized herbal medicine. Plant protoplasts can be enzymatically isolated from tissues culture. The composition of enzyme solutions for isolating protoplasts is critical to obtain high numbers of viable protoplasts. The length of incubation during the isolation process also determines the quantity and quality of the released protoplast. Each tissue source may require special conditions for successful isolation; therefore, the objective of this study was to observe the effect of enzyme solution and duration of incubation on the number of viable protoplasts released from P. angulata tissues in vitro. Five treatments for protoplast isolation combining cellulose and macerozyme were tested in vitro on leaf, stem and callus tissues as protoplast donors. Supplementing with an osmoticum (0.6 M mannitol) and a membrane stabilizer (10 mM CaCl2) provided significantly different numbers of viable protoplasts. In this study, all isolation conditions could not liberate protoplasts from callus tissues. The tested concentration of osmoticum only facilitated callus cells to plasmolyze. Spherical protoplasts containing distributed plastids were still inside the individual cells after 5 h incubation. Leaf protoplasts showed the most exciting results. The highest number of viable protoplasts (1.54 × 105 protoplasts/mL) was obtained from mesophyll tissues after 2 h incubation. The content of high-density green chloroplasts was a striking feature of leaf protoplasts. This feasible method to obtain leaf protoplasts from P. angulata is an excellent prospect for fusion experiments.Physalis angulata is a medicinal plant from the Solanaceae family. Utilization of tissue culture, especially protoplast techniques, has potential for supplying medicinal plants in the framework of their development as standardized herbal medicine. Plant protoplasts can be enzymatically isolated from tissues culture. The composition of enzyme solutions for isolating protoplasts is critical to obtain high numbers of viable protoplasts. The length of incubation during the isolation process also determines the quantity and quality of the released protoplast. Each tissue source may require special conditions for successful isolation; therefore, the objective of this study was to observe the effect of enzyme solution and duration of incubation on the number of viable protoplasts released from P. angulata tissues in vitro. Five treatments for protoplast isolation combining cellulose and macerozyme were tested in vitro on leaf, stem and callus tissues as protoplast donors. Supplementing with an osmoticum (0.6 M m...

The effect of tomato juices and bean sprout extracts on vitro shoot regeneration of Physalis angulata L.

Physalis angulata L. (Ciplukan) which belongs to Solanaceae is an important medicinal plant. In vitro culture medium contains carbon source, inorganic substance, vitamins, and plant growth regulators. However, organic growth supplements have frequently been added to improve regeneration capability of explants. This study was conducted to observe the effect of tomato juices and extract bean sprout on shoot regeneration and multiplication of in vitro nodal explants. The explants were cultured on MS basal medium + 6-benzyl amino purine (BAP) 2 mg/L + indole-3-acetic acid (IAA) 0.05 mg/L with and without organic supplements. Tomato juices (T) 5, 7.5 and 10% or bean sprout extract (B) 1.25, 2.5, and 3.75% were added as natural organic supplements. Almost all explants have produced shoots one week after culture. After six weeks of culture maximum shoot number (12.5±3.9) was produced in medium MS + T5 while maximum shoot length (10.7 ± 0.7 cm) was obtained in medium MS + T 7.5. Medium T tends to produce more sho...

The combination effect of auxin and cytokinin on in vitro callus formation of Physalis angulata L. – A medicinal plant

Physalis angulata L. (Ciplukan) is one member of Solanaceae that has a potential as herbal medicine. This plant grows wild in the crop fields, forest edges, etc. However, ciplukan is increasingly difficult to find recently. In vitro callus is an alternative source to produce secondary metabolite production as well as to regenerate plants through indirect organogenesis. This study aims to identify the response of hypocotyl explants on in vitro callus formation induced by a combination of auxin and cytokinins. Two types of cytokinins, Kinetin and BAP (0.5 ppm) were combined with three types of auxin, i.e. 2.4-D, IBA and IAA, at three concentrations 0.5, 1.0 and 1.5 ppm. In all combinations of cytokinin and auxin, 50–100% of hypocotyl explants derived from in vitro seedling were able to produce callus either in a compact or watery friable texture. In MS medium supplemented with 2.4-D, callus FW (fresh weight) began to decline in the fourth week after culture. Callus FW that increased until 5 weeks of culture...

Effect of Nitrogen Fertilizer on Calcium Oxalate Calibration Density of Porang Bulb (Amorphophallus muelleri Blume)

The aim of this research was to know the effects of nitrogen fertilizer to the shape and density of calcium oxalate (CaOx) crystals in porang tuber. Bulbil derived from porang plant in the first growth period seeded until the leaves fully open. Seedlings were transferred to a polybag after most of them has height 50 cm. After adapting for three weeks, the plants were treated with nitrogen fertilizer (urea) with a dose 0, 1, and 5 g/polybag. The plants were grown until the end of the growing period. The slides were prepared by slicing tuber as thin as possible using a sliding microtome. Sliced tuber of porang were cleared by using the modified clearing method. The slides were observed using light microscope to calculate the density of CaOx crystals. The densities of calcium oxalate crystals were analyzed using ANOVA (α = 0,05). The results showed that porang tuber has four kind of crystals i.e. raphide, druse, stiloid, and prism. Nitrogen fertilization had no significant effect on the densities of calcium oxalate crystals.

Calcium Oxalate Crystals (CaOx) on Porang (Amorphopallus muelleri Blume) Exposed and Unexposed Sun

Sunlight was suspected to affect CaOx crystal formation in porang . This research had aim to determine variations in forms of CaOx crystals and the influence of shade on the density of CaOx crystals in porang. Preparations for microscopic observation derived from sliced leaf, petiole, and tuber of porang that grown under shaded and exposed to sunlight conditions. Sliced organ was cleared by using the modified clearing method. The parameters that observed included shape, shape variety, and density of CaOx crystals. CaOx crystal density were analyzed using ANOVA followed by Tukey (α 0.05). The differences of crystal density between the edges and middle of the organ were analyzed using Paired Samples T Test. Microscopic observation showed that CaOx crystals were grouped into large (20-710 m) and small (1-15 m) crystals size. The density of CaOx crystals in plants exposed to sunlight was 3 times higher than the shaded plants. Leaf organ had the highest number of crystal compared to others organ. The tuber had the lowest density of CaOx crystals among organ. In addition, the shaded or exposed to sunlight condition had no effect on CaOx crystal density between the edges and center of the organ.