Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

Optimization and performance evaluation of microbial fuel cell by varying agar concentration using different salts in salt bridge medium

Purpose: Comparative study of various agar-agar (C14H24O9) percentage and different salts concentration in the salt bridge is carried out to check the efficiency of microbial fuel cell. Design/methodology/approach: Dual chambered microbial fuel cell was used for the overall experiments. Anode and cathode chambers were made of 500 ml plastic jar. Salt bridge was fabricated with agar-agar technical and 3 M NaCl in a PVC pipe of 2 cm long. Chemical Oxygen Demand, pH and electrical conductivity of wastewater were examined. Oxygen was supplied in the cathode chamber using the aquarium pump. Voltage (open circuit voltage) was observed using digital multimeter. Graphite rods were used as anode and cathode electrodes. Findings: Salt bridge was constructed of 3 M NaCl with 5, 7.5, 10 and 12 percent variation of agar amounts in MFC. The maximum outputs were observed 301, 306, 325 and 337.25 mV with the variation of agar 5, 7.5, 10 and 12 percentages respectively as well as chemical oxygen demand (COD) removal efficiency was observed 47.92, 56.25, 52.08 and 64.58 percentages respectively. The optimum agar concentration was found to be 12 percent and a maximum voltage of 337.25 mV and COD removal of 64.58 percent was achieved. After the optimization of agar percentage two salts i.e., Sodium chloride and potassium chloride were analysed. This study also reveals that the NaCl salt bridge is more efficient than KCl salt bridge for the same agar concentration. The maximum voltage for NaCl and KCl were 319 and 312 mV respectively. Research limitations/implications: The amount of electricity production is low and field scale implementation is difficult using microbial fuel cell. The research is still on progress in this field. Originality/value: here is very little research with salt bridge and MFC. Comparative study of different mole of salt is available but agar variation is not yet studied.

Stability of black grass jelly (Mesona chinensis) probioticated by encapsulation of Lactobacillus plantarum Mar8 with agar and gum Arabic

Wulandari NF, Yulinery T, Suharna N, Nurhidayat N. 2019. Stability of black grass jelly (Mesona chinensis) probioticated by encapsulation of Lactobacillus plantarum Mar8 with agar and gum Arabic. Nusantara Bioscience 11: 84-88. Black grass jelly [Mesona chinensis (Benth)] is one of the traditional food ingredients that can be used as the encapsulation material. However, syneresis easily occurs so that becomes a problem during storage. This study was conducted to evaluate the effect of type of stabilizers (agar and gum Arabic) and storage temperature (cold/4oC and ambient temperature/37oC) on black grass jelly stability and the viability of encapsulated Lactobacillus plantarum Mar8 in probiotication. The concentrations of stabilizers were 0.5%; 1.0%; 1.5% and 2.0% (w/v). The result showed that the syneresis rate was stable up to 2% of agar concentration at low storage temperature, while gum Arabic in the concentration of >1% resulted in increasing syneresis rate. Ambient temperature storage increased the syneresis rate. This study showed that agar or gum Arabic was supporting the probiotication as good encapsulant. Moreover, it was suitable for stabilizing or gelling agent at 0.5%-1% concentration to produce black grass jelly as RTD.

Cell Shape and Population Migration Are Distinct Steps ofProteus mirabilisSwarming That Are Decoupled on High-Percentage Agar

ABSTRACTSwarming on rigid surfaces requires movement of cells as individuals and as a group of cells. For the bacteriumProteus mirabilis, an individual cell can respond to a rigid surface by elongating and migrating over micrometer-scale distances. Cells can form groups of transiently aligned cells, and the collective population is capable of migrating over centimeter-scale distances. To address howP. mirabilispopulations swarm on rigid surfaces, we asked whether cell elongation and single-cell motility are coupled to population migration. We first measured the relationship between agar concentration (a proxy for surface rigidity), single-cell phenotypes, and swarm colony phenotypes. We find that cell elongation and single-cell motility are coupled with population migration on low-percentage hard agar (1% to 2.5%) and become decoupled on high-percentage hard agar (>2.5%). Next, we evaluate how disruptions in lipopolysaccharide (LPS), specifically the O-antigen components, affect responses to hard agar. We find that LPS is not essential for elongation and motility of individual cells, as predicted, and instead functions to broaden the range of agar concentrations on which cell elongation and motility are coupled with population migration. These findings demonstrate that cell elongation and motility are coupled with population migration under a permissive range of surface conditions; increasing agar concentration is sufficient to decouple these behaviors. Since swarm colonies cover greater distances when these steps are coupled than when they are not, these findings suggest that collective interactions amongP. mirabiliscells might be emerging as a colony expands outwards on rigid surfaces.IMPORTANCEHow surfaces influence cell size, cell-cell interactions, and population migration for robust swarmers likeP. mirabilisis not fully understood. Here, we have elucidated how cells change length along a spectrum of sizes that positively correlates with increases in agar concentration, regardless of population migration. Single-cell phenotypes can be decoupled from collective population migration simply by increasing agar concentration. A cell’s lipopolysaccharides function to broaden the range of agar conditions under which cell elongation and single-cell motility remain coupled with population migration. In eukaryotes, the physical environment, such as a surface matrix, can impact cell development, shape, and migration. These findings support the idea that rigid surfaces similarly act on swarming bacteria to impact cell shape, single-cell motility, and collective population migration.

The Effect of Sugar and Agar-agar Concentration on Nypa Fruit Slice Jam

Nypa fruticans or known as nypa produces considerable amount of fruit with high content of carbohydrate. The mesocarp of the mature fruit is a potential source of energy but underutilised. This research was conducted to study the production of nypa slice jam by focussing on the effect of sugar and agar-agar concentration on the physical, chemical and sensory properties of nypa slice jam. Sugar and agar-agar concentration affect rendemen, crude fibre content, total soluble solid, water content, colour, texture, taste, flavour and overal acceptance of slice jam from nypa fruit.

KAJIAN INDUKSI KALUS RUMPUT LAUT Kappaphycus alvarezii UNTUK PRODUKSI EMBRIO SOMATIK

Untuk mendukung program transgenesis pada rumput laut, embrio somatik dapat digunakan sebagai material untuk transfer gen baik secara individu sel ataupun kluster sel embriogenik, sehingga mempercepat keberhasilan dengan peluang transformasi yang lebih tinggi. Penelitian ini bertujuan untuk mengkaji induksi kalus rumput laut K. alvarezii untuk produksi sel embrio somatik (e.s.) dengan beberapa rasio zat pengatur tumbuh (ZPT) dan konsentrasi agar media induksi, sampai sel menjadi filamen. Penelitian terdiri atas dua tahap: Tahap (1) induksi kalus, dengan rasio ZPT asam indol asetat (IAA):kinetin = 0,5:0,0 mg/L; 1,0:1,0 mg/L; dan 2,0:0,2 mg/L dengan konsentrasi agar media induksi = 0,6%; 0,8%; 1,0%; dan 1,5%. Tahap (2) regenerasi massa sel e.s., dengan rasio IAA:kinetin = 0,1:1,0 mg/L; 0,0:0,1 mg/L dan tanpa ZPT dengan konsentrasi agar media = 0,4%; 0,6%; dan 0,8%. Untuk perkembangan sel-sel e.s. lebih lanjut dipelihara pada kultur cair. Hasil penelitian menunjukkan pada tahap induksi kalus, rasio IAA: kinetin = 1:1 mg/L dengan konsentrasi agar media 0,8% dan 1,0% menghasilkan persentase induksi kalus tertinggi (90%). Pada tahap regenerasi massa sel e.s., ZPT tidak berpengaruh terhadap perkembangan massa sel e.s., di mana tanpa ZPT dengan konsentrasi agar 0,6% memperlihatkan perkembangan tertinggi (rata-rata diameter massa sel 5 mm). Pada media cair, perkembangan sel e.s. dari single cell ukuran 3-4 mm menjadi filamen-filamen ukuran rata-rata 0,5 mm dapat dicapai dalam satu bulan kultur. Keberhasilan produksi sel e.s. K. alvarezii, selain sebagai material untuk transfer gen juga dapat dijadikan acuan dalam produksi benih rumput laut kultur jaringan.To support the program of seaweed transgenesis, somatic embryo can be used as a materials for gene transfer purpose either by individual or cluster of cells in accelerating the higher rate of transformation. This research aims to study the callus induction of seaweed K. alvarezii for production of somatic embrio (s.e) cell by different ratio of growth regulators (GR) and agar media concentrations. The study consists of two stages: stage (1) callus induction to the cells filament using GR of indol acetic acid (IAA):kinetin in ratio of 0.5:0.0 mg/L; 1.0:1.0 mg/L; and 2.0:0.2 mg/L on the media agar concentration of 0.6%; 0.8%; 1.0%; and 1.5%, and stage (2) regeneration of the cell mass of s.e, using GR of IAA:kinetin in ratio of 0.1:1.0 mg/L; 0.0:0.1 mg/L on the media agar concentration of 0.4%; 0.6%; and 0.8%. For further maintenance, the s.e cells were cultured in liquid media. The results of callus induction showed that the ratio of IAA:kinetin (1:1) on the media agar concentration of 0.8% and 1.0% produced the highest callus induction (90%). The study of mass cell regeneration of s.e showed did not different of GR IAA:kinetin ratio of the cell mass development, but the media agar concentration of 0.6% and 0.4% showed the higher growth of cell mass (in diameter size of 4-5 mm). The development of cells culture of s.e from the size of 3-4 mm to filament of 0.5 mm could be reached in one month of culture period. The success of K. alvarezii s.e production will be helpful not only as a material for gene transfer but also as a reference on tissue culture for seed production of seaweed.

Investigation of explants sterilization process, effect of light intensity and concentration of agar for callus induction of Kappaphycus alvarezii (Doty) doty (Rhodophyta) in vitro

Objective of this study was to ascertain the optimum condition for callus induction of K. alvarezii (Doty) in vitro such as to determine the explants sterilization process, the effect of intensity of light and the concentration of agar. Fresh thalli treated with 0.5% - 1% detergent for 5 mins followed by 0.5% - 1% betadine for 2 – 3 mins and incubated with 0.5% - 1% broad spectrum antibiotic mixture in PES medium for 1 day produced 95 – 98% bacteria free healthy explants. Two independent experiments with light intensity and agar contentration of the environment were carried out at 5 different levels of 0, 5, 25, 50, 70 µmol photon.m2.s-1 and 9 agar concentrations of 0.5%, 0.75%; 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.5%, 3.0%. The highest callus induction rate was (96 ± 3.5 – 98 ± 2.1%) at 5 - 25 µmol photon.m-2.s-1 and (87 ± 5.8% – 90 ± 5.0%) in 1% - 3% agar concentration after 2 weeks of explants. The highest callus living rate was 98% at the light intensity of 25 µmol photon.m-2.s-1 and (75 ± 5.7 – 84 ± 1.1%) in 0.75 – 1.5% agar concentration after 2 months of explants. The highest callus re-induction rate was 50 – 55% at the light intensity of 5 – 25 µmol photon.m-2.s-1 and 60 – 65% in 1 – 1.5% agar concentration. Callus was not observed in dark condition (0 µmol photon.m-2.s-1). These calluses, that were strong, big and had filamentous type, will be a good material for the next production stage of embryonic callus production and seedling regeneration from micropropagules.