Inhibitory effect of kanamycin on in vitro culture of Lycopersicon esculentum Mill cv. Mt11.

Excised cotyledons of tomato (Lycopersicon esculentum Mill cv. MT11) were cultured on selective medium containing kanamycin at various concentrations (50, 100, 200, 300 mg/L). Significant toxic effects were observed when the cotyledon explants were grown on MS medium supplemented with 5mg/L kinetin and 100 mg/L kanamycin. The regeneration of callus was decreased as the concentration of kanamycin increased from 200 to 300 mg/L. Explants grown on MS medium supplemented with 5mg/L kinetin and 50 mg/L kanamycin showed the least toxic effects (mean survival rate 48.0% ± 0.19) compared to the rest of the concentrations tested. Even though 100 mg/L of kanamycin allows the non-transformed explants to grow on the medium, the shoot primordia would not develop further.The result suggests that 100 mg/L of kanamycin can be used effectively to differentiate between non-transformed and transformed MT11 tomato explants with a death rate of more than 82% of non-transformed explants, after 4 weeks of incubation on selection medium. Therefore, 100 mg/L kanamycin is suitable for minimal inhibition concentration for MT11 and true transformants can be selected at this concentration for the transformation system

Protein expression of Late Elongated Hypocotyl (LHY) homolog genes of teak in Escherichia coli

Expression of an isolated gene in a system that directly translates it into a protein is an important step to study the protein encoded by the gene. The isolated gene can be expressed in vivo by a heterologous system. In this study, a bacteria system was used to translate the Tectona grandis Late Elongated Hypocotyl (Tg-LHY) gene, which was isolated from flowering tissues of teak (Tectona grandis). The gene was cloned into the pET 14b vector (Novagen) and transformed into BL 21(DE3)/pLysS and Rosetta 2 expression host cells (Novagen). Rosetta 2 host cell has been found to be a good candidate to express the Tg-LHY protein from plant origin, as it recognizes the codon that was found in plant but rarely used in bacteria. The expressed protein was about an expected size, which was 90 kD. Western blot analysis using antibody against His-tag, which was fused to the Tg-LHY protein, proved that the expressed protein was Tg-LHY protein

Biological parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) embryogenic calli via microprojectile bombardment

Biological parameters affecting microprojectile bombardment delivery of DNA into oil palm embryogenic calli were optimized by monitoring transient GUS gene expression. The parameters optimized were the following; explant type using gold and tungsten microcarrier, bombardment preculture, time between bombardment and GUS staining, genotype, immature embryo preculture, DNA concentration, osmoticum type and concentration, and osmoticum treatment duration before and after bombardment. An independent experiment was carried out to study the effect of each parameter and its variables on transient GUS expression. ANOVA was carried out for each experiment using completely randomized design and the treatment means were compared using Duncan's Multiple Range Test. The highest transient GUS expression was observed 2 days post-bombardment using embryogenic calli derived from immature embryos. Bombardment was carried out using 300 μg of gold microcarriers coated with 1.5 μg of DNA 24 h after transfer to fresh medium. GUS expression could be further enhanced when calli were transferred to medium containing osmoticum (0.4 M mannitol) 2 h prior to bombardment. Highly significant differences between the variables were observed for all the parameters studied except genotype

Characterisation of Bifidobacterium species—a review

Identification of Bifidobacterium species are a difficult task because of phenotypic and genetic heterogeneities. Various DNA-based techniques to rapidly characterise Bifidobacterium species and to support the conventional biochemical and morphological classification methods have been described. Sequencing of the 16S rRNA gene and 16S to 23S internally transcribed spacer region and comparing with the sequences data present in GenBank are the most popular techniques in identifying Bifidobacterium species. Conserved sequences other than the 16S rRNA gene such as ldh, recA and hsp60 genes have become worthy tools for the elucidation of various taxonomic features such as genera, species and strains of Bifidobacterium. However, as an alternative to sequencing which is both time consuming and technically demanding, genus- or species-specific primers or probes were successfully designed to rapidly identify Bifidobacterium species. In this review, amplified ribosomal DNA restriction analysis (ARDRA) method derived from the 16S rRNA gene is also discussed because of it rapid, reproducible and easy to handle characteristics. Furthermore, randomly amplified polymorphic DNA (RAPD), Pulsed-Field Gel Electrophoresis (PFGE) and repetitive elements fingerprinting (Rep) were the popular methods to study the genetic diversity among Bifidobacterium species due to its versatility

Aquilaria malaccensis polyploids as improved planting materials

Aquilaria malaccensis is an agarwood-producing timber species used in many traditional remedies and modern therapeutic treatments and perfume industries. In this study, we aimed to enhance A. malaccensis phytochemical content through in-vitro polyploidisation. Shoot tip and nodal segment from 8-week-old in-vitro A. malaccensis plantlets were treated with different concentrations of colchicine and trifluralin at various exposure times to obtain polyploids. Tetraploid plantlets (10%) was obtained using nodal segment explants treated with 0.1 mM trifluralin at 120 hours. Chemical profiling of diploid and tetraploid samples (leaf, stem and root) was evaluated separately using headspace-solid phase microextraction (HS-SPME) combined with gas chromatograph mass spectrometry (GCMS). Phytochemical content increased in tetraploid, particularly in stem whereby the total phytochemical contents were 43.19% in tetraploid compared with 5.87% in diploid. The HS-SPME-GCMS analyses showed that tetraploid stem contained high levels of sesquiterpenoids found in agarwood oil such as α-eudesmol (18.3%), α-gurjunene (8.61%) and γ-gurjunene (6.22%). On the other hand, aromadendrene (2.49%) and α-humulene (3.38%) were detected in diploid samples. Tetraploid leaf samples were observed to contain α-humulene (3.79%) while diploid only contained (2E) tridecenol (19%). There were no significant differences between diploid and tetraploid in terms of total phytochemical content in root samples. Nevertheless, high sesquiterpenoid content, γ-gurjunene (14.0%), was detected in tetraploid sample while γ-muurolene (2.96%), in diploid. α-Guaiene content was higher in root samples of diploid (6.49%) than tetraploid (1.09%). These results demonstrated that tetraploid plantlets led to higher yield of total phytochemical content and might facilitate production of high quality A. malaccensis clones

Assessment of hairy roots induction in Solenostemon scutellarioides leaves by different strains of Agrobacterium rhizogenes

Hairy roots of Solenostemon scutellarioides were induced by inoculation of leaf explants with Agrobacterium rhizogenes strains TR 105, LBA 9402, 8196 and ATCC 15834. These strains showed different abilities to induce hairy root formation on the leaf explants. Assessment of the plant’s susceptibility to the different strains of A. rhizogenes showed that strains ATCC 15834, TR 105, LBA 9402, and 8196 produced 56.3, 25.5, 21.5 and 13.8% transformation efficiencies, respectively. Acetosyringone was found to be useful for the enhancement of hairy roots formation in S. scutellarioides

Optimization of submerged culture conditions for the production of mycelial biomass and exopolysaccharides from lignosus rhinocerus

Tiger’s Milk mushroom (Lignosus rhinocerus) is a highly priced medicinal mushroom utilized in traditional medicine to treat various diseases. However, due to insufficient wild L. rhinocerus, submerged culture conditions and nutritional requirements for the production of mycelial biomass and exopolysaccharide (EPS) from L. rhinocerus were studied using one-factor-at-a-time and orthogonal matrix method in shake flask culture. The optimal pH and temperature for ideal production of mycelial biomass and EPS were found to be at pH6 and 25°C, respectively. The optimal compositions for mycelial biomass production were 80 g/L of glucose, 4 g/L of potassium nitrate, 0.4 g/L of FeSO4.7H2O and 0.1 g/L of CaCI2. Subsequently, the optimal compositions for EPS production were 80 g/L of glucose, 4 g/L of potassium nitrate, 1.4 g/L of FeSO4.7H2O and 1.1 g/L of CaCI2. The maximum mycelial biomass and EPS concentrations achieved in a 1.5 L stirred-tank bioreactor were 6.3788 g/L and 1.2 g/L, respectively. Mycelial biomass production was about 3 times higher than that at the basal medium. However, EPS production indicated no significant difference at the basal medium. In addition, the concentrations for α-amylase, β-amylase, cellulase and invertase in optimal medium were 2.87, 1.07, 3.0 and 3.0 mg/mL, respectively. Current findings suggest that the production of mycelial biomass and EPS of L. rhinocerus can be enhanced dramatically by controlling the culture conditions and modifying the medium’s composition

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

Isolation and characterization of LHY homolog gene expressed in flowering tissues of Tectona grandis (teak)

Floral initiation of teak through molecular biology approach is being studied for better understanding of teak flower development. Through PCR subtractive hybridization method, LHY homolog gene has been isolated from teak flowering tissues. The full-length cDNA of the gene was 2948 base pair (bp) and potentially encoded for 768 amino acids. It was named Tectona grandis LHY (Tg-LHY), as the gene was similar to the LHY gene of some species. Amino acid sequence alignment revealed that Tg-LHY was similar to LHY of Castanea sativa, LHY ofPhaseolus vulgaris and LHY of Arabidopsis thaliana. The highly conserved region found in Tg-LHY was the MYB protein, which is the DNA-binding protein responsible in negative feedback loop reaction of central oscillator in plant circadian clock system. The level of gene expression was found to be high four hours after dawn in flowering shoots and flower. This paper reported the isolation and characterization of the gene