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Masani M.Y.A.,Malaysian Palm Oil Board | Noll G.A.,University of Munster | Parveez G.K.A.,Malaysian Palm Oil Board | Sambanthamurthi R.,Malaysian Palm Oil Board | And 2 more authors.
PLoS ONE | Year: 2014

Background: Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. Methodology/Principal Findings: We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. Conclusions/Significance: We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants. © 2014 Masani et al. Source

Munt O.,University of Munster | Arias M.,Neiker | Hernandez M.,Neiker | Ritter E.,Neiker | And 3 more authors.
Industrial Crops and Products | Year: 2012

Taraxacum brevicorniculatum produces high-quality natural rubber in its roots and could be developed as an alternative commercial source of this valuable raw material. However, current wild type accessions have a low biomass and branched roots that make them difficult to harvest. We set out to determine the optimum fertilizer and spacing requirements for T. brevicorniculatum plants in greenhouse and field trials, aiming to increase root biomass and reduce branching thus maximizing rubber yield and reducing losses during harvesting. Our preliminary data from greenhouse trials show that fertilizers containing calcium and magnesium in addition to NPK (nitrogen, phosphorus and potassium) increase root biomass by 25% compared to standard NPK fertilizer plus boron, and by 15% compared to NPK plus calcium. Fertilizers with a low N:P ratio increased the root biomass in greenhouse and field trials. Dense sowing (5-10. cm between plants) inhibits root branching and leads to the formation of single taproots that can be harvested easily. We therefore provisionally recommend the application of low N:P ratio NPK fertilizer supplemented with calcium and magnesium to closely-spaced plants in order to achieve the greatest increase in plant biomass per hectare. © 2011 Elsevier B.V. Source

Masani M.Y.A.,Malaysian Palm Oil Board | Noll G.,University of Munster | Parveez G.K.A.,Malaysian Palm Oil Board | Sambanthamurthi R.,Malaysian Palm Oil Board | And 2 more authors.
Plant Science | Year: 2013

Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×106 per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10μM naphthalene acetic acid, 2μM 2,4-dichlorophenoxyacetic acid, 2μM indole-3-butyric acid, 2μM gibberellic acid and 2μM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid. © 2013 Elsevier Ireland Ltd. Source

Schmidt T.,University of Munster | Hillebrand A.,University of Munster | Wurbs D.,University of Munster | Wahler D.,University of Munster | And 4 more authors.
Plant Molecular Biology Reporter | Year: 2010

Rubber biosynthesis requires the action of specific enzymes known as cis-prenyltransferases (CPTs). These enzymes are responsible for the sequential addition of isopentenyl pyrophosphate units to the growing polyisoprene chain, a biochemical reaction thought to be stimulated by the presence of small rubber particle proteins (SRPPs). We have cloned, characterized, and analyzed the expression of three CPT genes (TkCPT1-3) and five SRPP genes (TkSRPP1-5) from the rubber-producing plant Taraxacum koksaghyz. The deduced TkCPT amino acid sequences showed significant levels of sequence identity with Hevea brasiliensis CPTs. We also found no obvious differences between SRPPs from T. koksaghyz, another rubber producer, and a non-rubber plant. The roles of the individual TkCPTs and TkSRRPs in rubber biosynthesis are discussed. © Springer-Verlag 2009. Source

Van Deenen N.,University of Munster | Bachmann A.L.,University of Munster | Schmidt T.,University of Munster | Schaller H.,University of Strasbourg | And 3 more authors.
Molecular Biology Reports | Year: 2012

Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products. © Springer Science+Business Media B.V. 2011. Source

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