Optimization of Parameters Involved in the Transformation of Oil Palm Using the Biolistic Method

Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device have been optimized. The physical parameters tested were : helium pressure, distance from rupture disc to the macro carrier, distance from macro carrier to the stopping plate, distance from stopping plate to the target tissue, vacuum pressure, number of bombardments, particle types and sizes, and the effect of calcium chloride and spermidine on microcarrier-DNA binding. The optimized biological parameters were: explant types with gold micro carrier, explant types with tungsten, duration of callus culture in fresh medium prior to bombardment, duration between bombardment and GUS staining, genotype, immature embryo preculture duration, DNA concentration, osmoticum type and concentration and osmoticum treatment duration before and after bombardment. Independent experiments were carried out to study the effects of each parameter and its variables on transient expression. Two days after bombardment, the tissues were stained with GUS assay buffer for 16-20 hours at 37°C and the blue spots counted under a binocular microscope. All the variables used in these experiments were found to be significantly different except for vacuum pressure, bombardment number and genotype. The efficiency of GUS gene expression was measured in embryogenic calli and young leaves of mature and seedling palms using five constructs carrying different promoters : Emu; Ubil; Actl, 35S and Adhl were evaluated to identify the most suitable promoter for use in oil palm. The GUS gene expression from the different promoters was assayed histochemically and fluorometrically from a total of 200 plates of target tissues in eight independent experiments. Significant effects on transient GUS gene expression were demonstrated by each of the different promoters tested. The effectiveness of kanamycin; geneticin (G-418); neomycin, hygromycin and basta as selection agents to inhibit growth of oil palm embryogenic calli was evaluated. Embryogenic calli were separately exposed to all these selection agents at different concentrations ranging from 1 to 2000 mg/l for a period of one month. This was done in two replicates and repeated twice to ensure reproducibility of the selection system. Of the five compounds tested, hygromycin and basta were found to be most suitable as selection agents for oil palm as they can stop the growth of embryogenic calli at lower concentrations.Bombarded embryogenic calli were exposed to 40 or 80mg/l of selective agents after 1 or 3 weeks. It was found that there were no significant differences in the number of resistant embryogenic calli produced per plate when selected at different concentrations and time. The presence of transgenes in the resistant embryogenic calli was confirmed by PCR and Southern analysis. Transgenic embryogenic calli were later regenerated into whole plants and their transgenic status verified by PCR and Southern analysis. Problems faced during the study and their solutions are also discussed. As oil palm has a long breeding cycle, inheritance of transgenes cannot be demonstrated within the period of this study. Therefore, rice, a model crop for monocot transformation, was also used for transformation experiments. Calli derived from immature embryos were bombarded and were selected on hygromycin. Resistant calli isolated were regenerated into whole plants. Two transgenic lines were obtained. T1 and T2 from one of the clones were also produced and analysed. Integration and inheritance of the transgenes were followed by phenotypic and genotypic analysis

Transgenic plants producing polyhydroxyalkanoates

Currently, the polyhydroxybutyrate (PHB) copolymer, polyhydroxy-co-valerate (PHBV) is being produced commercially using a two-stage glucose / propionate fed batch fermentation process using Ralstonia eutropha. The economics of the manufacturing process are still a major barrier to the widespread use of polyhydroxyalkanoates (PHAs) and intensive efforts are being made to reduce the cost of production by means of bioprocess design and metabolic engineering of production strains. However, despite these improvements, the production costs are still high compared to petroleum-derived commodity plastics. An alternative strategy for lowering production costs that has been proposed is to develop transgenic plants that produce PHAs. This strategy is considerably cheaper because the PHAs production from plants does not require expensive fermentation equipment and processing facilities

Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter

One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (β-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (β-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation

A Study on Relation between Self Concept and Academic Achievement among Secondary School Students of Jammu District

Self-concept is the whole set of attitudes, opinions, and cognitions that a person has of himself while the academic achievement or (academic) performance is the outcome of education - the extent to which a student, teacher or institution has achieved their educational goals. The objective of the paper is to study the relation between the self concept and academic achievement i.e is there any relation between these two variables or not. The paper is based the sample of 248 students taken from the senior secondary schools of Jammu district through the random sampling technique out of 9 selected schools. However the findings of the study show the significant relation between the self concept and academic achievement. Keywords: self concept, academic achievement, relation, schools, samplin

Effect of Indian Jujube pulp on physico-chemical and sensory characteristics of chicken sausages

Abstract A study was undertaken to evaluate the effect of Indian jujube pulp on the quality attributes of chicken sausages incorporated at 0%, 5%, 10% and 15% level (replacing lean meat) into the formulation of chicken sausages. The proximate, physico-chemical and sensory properties of sausages were analyzed. Among the different proximate and physicochemical parameters, a significant (p<0.05) decrease in pH, emulsion stability, ash content, cooking yield, ether extract as well as crude protein percentage with each subsequent incorporation level was accompanied by a gradual increase in moisture of cooked sausages. However, crude fiber content increased significantly (p<0.05) with increasing level of inclusion. The sausages with 10% level of Indian jujube pulp incorporation had highest (p<0.05) overall acceptability score

The feasibility of producing oil palm with altered lignin content to control Ganoderma disease

Oil palm is a major crop which is grown for the production of vegetable oil used in foods, cosmetics and biodiesel. The palm is of major economic importance in southeast Asia where it is grown extensively in Malaysia and Indonesia. There is concern about Ganoderma rots of oil palm which need to be controlled to prevent major infection. However, the basic mechanism of white-rot infection has been ignored. White rot implies that fungi attack the lignin component of woody tissue leaving the white cellulose exposed. The fungus grows within palms by utilizing cellulose in the tree. By altering the lignin fraction of oil palm losses may be reduced. Methods for altering lignin in plants are reviewed here to indicate how similar transformations could be attempted for oil palm. In addition, progress in transforming oil palm is described. Lignin is extremely complex and this may explain why it has not been studied in oil palm. Some crops transformed with Bacillus thuringiensis toxin genes have increased lignin and modified oil palm have been produced using B. thuringiensis genes. These require to be tested for lignin concentration and structure. The nomenclature of the disease organism is discussed. The prospects for altered lignin oil palm are considered herein.Fundação para a Ciência e a Tecnologia (FCT) - SFRH ⁄ BPD ⁄ 34879 ⁄ 200

IMPACT OF PACLOBUTRAZOL ON THE GROWTH AND DEVELOPMENT OF NURSERY GROWN CLONAL OIL PALM (Elaeis guineensis Jacq.)

The economic life of an oil palm (Elaeis guineensis Jacq.) plant is associated with the stature of the palm as the height of each oil palm affects the cost of harvesting. Shorter palm allows ease of harvesting and better fruit recovery hence contributes to the increase of the fresh fruit bunch (FFB) yield. This study was conducted to investigate the effect of paclobutrazol (PBZ), an inhibitor of gibberellin (GA) biosynthesis, on the growth and development of clonal oil palms maintained under nursery conditions. Application of PBZ significantly reduced the growth of clonal palms with the optimal concentration of PBZ being 50 mg litre-1 when applied as a foliar spray. PBZ-treated palms exhibited shorter fronds that were dark green in colour and plants had a more compact structure as compared to untreated controls. Longitudinal sections of PBZ-treated oil palm clones revealed that both leaves and stems comprised of fewer cells each with a smaller volume. PBZ-treated plants exhibited a higher rate of photosynthesis compared to controls and this was correlated with an accumulation of starch in stem cells. These results provide evidence that the vegetative growth of oil palm is regulated by GA levels and that by manipulating the concentration of these hormones it may be possible to attenuate the oil palm height and ultimately increase productivity

Factors affecting delivery and transient expression of β-glucuronidase gene in Dendrobium Sonia protocorm-like-body

The effect of the biolistic device parameters and other factors affecting delivery and expression of uidA gene in Dendrobium Sonia was investigated. Three week old protocorm like body (PLB) were bombarded with gold microparticles coated with pAHC25 plasmid harbouring the uidA gene which encodes β-glucuronidase. The factors investigated were the helium pressure, target distance, macrocarrier flight distance to stopping screen, distance from stopping screen to target tissues, vacuum pressure, gold microparticles size, spermidine and calcium on DNA precipitation, and the number of bombardments. Two days after bombardment, the PLB were subjected to histochemical GUS assay, and transient GUS activity was recorded as blue spots using a Leica stereomicroscope. All the factors tested showed significant effects (p<0.05) on the delivery of DNA and expression of the uidA gene in Dendrobium PLB except for calcium. Surviving PLBs were able to grow and regenerate normally into plantlets.Keywords: biolistic transformation, orchid, Dendrobium, protocorm like body, -glucuronidase assa

Resistência microbiana a drogas em linhagens de Escherichia coli isoladas de fontes alimentares

A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in medicine and agriculture. Antibiotic resistance in Escherichia coli is of particular concern because it is the most common Gram-negative pathogen in humans. Hence this study was conducted to determine the antibiotic sensitivity pattern of E. coli isolated from different types of food items collected randomly from twelve localities of Hyderabad, India. A total of 150 samples comprising; vegetable salad, raw egg-surface, raw chicken, unpasteurized milk, and raw meat were processed microbiologically to isolate E. coli and to study their antibiotic susceptibility pattern by the Kirby-Bauer method. The highest percentages of drug resistance in isolates of E. coli were detected from raw chicken (23.3%) followed by vegetable salad (20%), raw meat (13.3%), raw egg-surface (10%) and unpasteurized milk (6.7%). The overall incidence of drug resistant E. coli was 14.7%. A total of six (4%) Extended Spectrum β-Lactamase (ESBL) producers were detected, two each from vegetable salads and raw chicken, and one each from raw egg-surface and raw meat. Multidrug resistant strains of E. coli are a matter of concern as resistance genes are easily transferable to other strains. Pathogen cycling through food is very common and might pose a potential health risk to the consumer. Therefore, in order to avoid this, good hygienic practices are necessary in the abattoirs to prevent contamination of cattle and poultry products with intestinal content as well as forbidding the use of untreated sewage in irrigating vegetables.Variedade de alimentos e fontes ambientais contem bactérias resistentes a uma ou mais drogas antimicrobianas usadas em medicina e agricultura. Resistência antibiótica pela Escherichia coli é particularmente preocupante porque ela é o patógeno mais comum Gram negativo em humanos. Portanto este estudo foi conduzido para determinar o aspecto de sensibilidade antibiótica da E. coli isolados de diferentes tipos de alimentos obtidos ao acaso de 12 localidades de Hyderabad, India. Um total de 150 amostras compreendendo saladas, vegetais, superfícies de ovos crus, galinhas cruas, leite não pasteurizado e carne crua foram processados microbiologicamente para isolar E. coli e estudar o quadro de sensibilidade antibiótica pelo método de Kirby-Bauer. A maior percentagem de resistência à droga foi isolada de E. coli obtidos de galinha crua (23,3%) seguido de saladas e vegetais (20%), carne crua (13,3%), superfície do ovo cru (10%) e leite não pasteurizado (6,7%). Incidência total de E. coli resistente foi de 14,7%. Um total de seis (4%) Extended Spectrum β-Lactamase (ESBL) produtores foram detectados, dois cada de salada de vegetais e galinha crua e um cada de superfície de ovo cru e carne crua. Espécies resistentes a múltiplas drogas de E. coli são matéria de preocupação uma vez que os genes de resistência podem facilmente ser transferidos para outras linhagens. O ciclo do patógeno é muito comum nos alimentos e pode ser risco potencial para a saúde do consumidor. Portanto, para evitar isto boas práticas de higiene são necessárias nos abatedouros para prevenir a contaminação de gado e aves com conteúdo intestinal assim como proibir o uso de águas de esgoto não tratadas para irrigar vegetais