Showing papers in "Plant Cell Reports in 2003"
TL;DR: A novel model for IAA signaling pathway mediated by extracellular ROS produced by cell-wall POXs is proposed and possible controls of the IAA-POX reactions by a fungal alkaloid are discussed.
Abstract: Extracellularly secreted plant peroxidases (POXs) are considered to catalyze the generation of reactive oxygen species (ROS) coupled to oxidation of plant hormone indole-3-acetic acid (IAA) and defense-related compounds salicylic acid (SA), aromatic monoamines (AMAs) and chitooligosaccharides (COSs). This review article consists of two parts, which describe H2O2-dependent and H2O2-independent mechanisms for ROS generation, respectively. Recent studies have shown that plant POXs oxidize SA, AMAs and COSs in the presence of H2O2 via a conventional POX cycle, yielding the corresponding radical species, such as SA free radicals. These radical species may react with oxygen, and superoxide (O2·−) is produced. Through the series of reactions 2 moles of O2·− can be formed from 1 moles of H2O2, thus leading to oxidative burst. It has been revealed that the ROS induced by SA, AMAs and COSs triggers the increase in cytosolic Ca2+ concentration. Actually POXs transduce the extracellular signals into the redox signals that eventually stimulate the intracellular Ca2+ signaling required for induction of defense responses. On the other hand, IAA can react with oxygen and plant POXs in the absence of H2O2, by forming the ternary complex enzyme-IAA-O2, which readily dissociates into enzyme, IAA radicals and O2·−. This article covers the recent reports showing that extracellularly produced hydroxy radicals derived from O2·− mediate the IAA-induced cell elongation. Here a novel model for IAA signaling pathway mediated by extracellular ROS produced by cell-wall POXs is proposed. In addition, possible controls of the IAA-POX reactions by a fungal alkaloid are discussed.
516 citations
TL;DR: Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios, and allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties.
Abstract: The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (β-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3%. Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios.
239 citations
TL;DR: A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the AgRobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat.
Abstract: An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study—selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50–80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of the aroA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.
180 citations
TL;DR: An efficient protocol for the production of transgenic Brassica napus cv.
Abstract: An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.
173 citations
TL;DR: These findings provide the first report of simultaneous shoot organogenesis and somatic embryogenesis of African violet explants in response to TDZ.
Abstract: Regeneration via shoot organogenesis and somatic embryogenesis was observed from thidiazuron (TDZ)-treated leaf and petiole explants of greenhouse- and in vitro-grown African violet plants The response of cultures to other growth regulators over a range of 05 µM to 10 µM was 50% less than that observed with TDZ A comparative study among several cultivars of African violet indicated that 'Benjamin' and 'William' had the highest regeneration potential In 'Benjamin', higher frequencies of shoot organogenesis (twofold) and somatic embryogenesis (a 50% increase) were observed from in vitro- and greenhouse-grown plants, respectively At concentrations lower than 25 µM, TDZ induced shoot organogenesis, whereas at higher doses (5–10 µM) somatic embryos were formed These findings provide the first report of simultaneous shoot organogenesis and somatic embryogenesis of African violet explants in response to TDZ
156 citations
TL;DR: High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants.
Abstract: The objective of the present research was to introduce genes with antifungal potential into the commercially important apple cvs Elstar
and Holsteiner Cox in order to establish resistance against fungal diseases The gene encoding the stilbene synthase
(Vst1) from Vitis vinifera L, responsible for the synthesis of the phytoalexin resveratrol in grapevine, and the gene for a polygalacturonase-inhibiting protein (PGIP) from kiwi (Actinidia deliciosa) were transferred into Holsteiner Cox
and Elstar via Agrobacterium tumefaciens-mediated transformation A total of nine transgenic Holsteiner Cox clones and one transgenic E clone carrying the stilbene-synthase gene as well as three transgenic Holsteiner Cox lines harbouring the polygalacturonase-inhibiting protein from Kiwi were identified via polymerase chain reaction and Southern blot analysis High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants
140 citations
TL;DR: Results prove successful expression of the entire PHB pathway in plastids, concomitant, however, with growth deficiency and male sterility.
Abstract: The pathway for synthesis of polyhydroxybutyrate (PHB), a polyester produced by three bacterial enzymes, was transferred to the tobacco plastid genome by the biolistic transformation method. The polycistronic phb operon encoding this biosynthetic pathway was cloned into plastome transformation vectors. Following selection and regeneration, the content and structure of plant-produced hydroxybutyrate was analysed by gas chromatography. Significant PHB synthesis was limited to the early stages of in vitro culture. Within the transformants, PHB synthesis levels were highly variable. In the early regeneration stage, single regenerates reached up to 1.7% PHB in dry weight. At least 70% of plant-produced hydroxybutyric acid was proven to be polymer with a molecular mass of up to 2,500 kDa. PHB synthesis levels of the transplastomic lines were decreasing when grown autotrophically but their phb transcription levels remained stable. Transcription of the three genes is divided into two transcripts with phbB being transcribed separately from phbC and phbA. In mature plants even low amounts of PHB were associated with male sterility. Fertility was only observed in a mutant carrying a defective phb operon. These results prove successful expression of the entire PHB pathway in plastids, concomitant, however, with growth deficiency and male sterility.
136 citations
TL;DR: In this procedure, about 33% of globular embryos not only developed into the cotyledonary stage but rooted simultaneously, eliminating a separate rooting step, and significant improvement in embryogenesis was achieved.
Abstract: A highly efficient somatic embryo production and maturation procedure has been developed to regenerate plantlets from cotton (Gossypium hirsutum). This procedure involves the acceleration of differentiation through manipulations of nutrient and microenvironment conditions. Embryogenic calli, initiated from hypocotyls or cotyledonary leaf sections on MS medium containing 0.1 mg/l 2,4 dichlorophenoxyacetic acid, 0.5 mg/l kinetin, and 3% maltose produced globular-stage somatic embryos when transferred to hormone-free MS medium supplemented with high concentrations of nitrate. Subculture of globular embryos on hormone-free MS medium led to the development of torpedo- and cotyledonary-stage at a low frequency (two to four per plate) with the majority of embryos lacking further growth or entering into the dedifferentiation stage. Significant improvement in embryogenesis (two- to threefold) was achieved when calli were cultured on 1/5-strength MS medium irrespective of stress treatment. However, the frequency of globular embryos developing into normal plantlets improved considerably (20–24 per plate) when cultured on filter paper placed on MS medium. In this procedure, about 33% of globular embryos not only developed into the cotyledonary stage but rooted simultaneously, eliminating a separate rooting step. More than 70% of cotyledonary embryos developed into normal plantlets when cultured on full- strength MS medium containing 0.05 mg/l gibberellic acid.
127 citations
TL;DR: Flow cytometric analysis showed that the micropropagation protocol followed here did not affect the ploidy level of somatic embryo-derived plantlets, and was found to stimulate embryo germination.
Abstract: An improved protocol is described for the large-scale micropropagation of an elite date palm (Phoenix dactylifera L.) cultivar, Deglet Nour. Clonal plants were regenerated from somatic embryos derived from highly proliferating suspension cultures. Friable embryogenic calli were initiated from both leaf and inflorescence explants. Suspension cultures consisting of pro-embryonic masses were established from calli showing a high competency for somatic embryogenesis. The subculture of suspensions in liquid medium enriched with low amounts of plant growth regulators (1 mg l-1 2,4-dichlorophenoxyacetic acid with 300 mg l-1 charcoal) resulted in the differentiation of large numbers of somatic embryos. The productivity of the cultures increased 20-fold (from 10 to 200 embryos per month per 100 mg fresh weight of embryogenic callus) when embryogenic suspensions were used instead of standard cultures on solid media. The overall production of somatic embryos reached 10,000 units per litre per month. Partial desiccation of the mature somatic embryos, corresponding to a decrease in water content from 90% to 75%, significantly improved germination rates (from 25% to 80%). The cutting back of the cotyledonary leaf was also found to stimulate embryo germination. Flow cytometric analysis showed that the micropropagation protocol followed here did not affect the ploidy level of somatic embryo-derived plantlets.
126 citations
TL;DR: A reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata was developed and the survival and germination rates of encapsulated embryos increased following storage at 4°C, which could be useful for the rapid clonal propagation and dissemination of synthetic seed material.
Abstract: We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, α-naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l-1 casein hydrolysate and 10 mg l-1 TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 mM CaCl2. A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4°C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4°C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata.
124 citations
TL;DR: Observations support the conclusion that even with the new maturation medium somatic embryos grow approximately only halfway through the normal sequence of development and then prematurely discontinue growth.
Abstract: Clonal production of loblolly pine (Pinus taeda L) through somatic embryogenesis has the potential to meet the increasing industrial demands for high-quality uniform raw materials A major barrier to the commercialization of this technology is the low quality of the resulting embryos Twenty-five newly initiated loblolly pine genotypes were followed through the process of liquid culture establishment, embryo maturation, germination, and retrieval from cryogenic storage A maturation medium, capable of promoting the development of loblolly pine somatic embryos that can germinate, is presented that combines 1/2 P6 modified salts, 2% maltose, 13% polyethylene glycol 8000 (PEG), 5 mg/l abscisic acid (ABA), and 25 g/l Gelrite A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also described A set of somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed The quality of the resulting embryos was examined and compared to that of zygotic embryos using such parameters as morphology, dry weight, germination performance, and gene expression All of the observations that were made support the conclusion that even with the new maturation medium somatic embryos grow approximately only halfway through the normal sequence of development and then prematurely discontinue growth
TL;DR: Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog medium to make the protocol economically advantageous.
Abstract: Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l–1 N6-benzylaminopurine (BAP) and 0.5 mg l–1 indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l–1 BAP and 0.5 mg l–1 kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l–1 naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l–1) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.
TL;DR: The high frequency of plant regrowth from alginate-coated micropropagules coupled with high viability percentage after 28 days of storage is highly encouraging for the exchange of cassava genetic resources.
Abstract: We report the encapsulation of in vitro-derived nodal cuttings or shoot tips of cassava in 3% calcium alginate for storage and germplasm exchange purposes. Shoot regrowth was not significantly affected by the concentration of sucrose in the alginate matrix while root formation was. In contrast, increasing the sucrose concentration in the calcium chloride polymerisation medium significantly reduced regrowth from encapsulated nodal cuttings of accession TME 60444. Supplementing the alginate matrix with increased concentrations of 6-benzylaminopurine and α-naphthaleneacetic acid enhanced complete plant regrowth within 2 weeks. Furthermore, plant regrowth by encapsulated nodal cuttings and shoot tips was significantly affected by the duration of the storage period as shoot recovery decreased from almost 100% to 73.3% for encapsulated nodal cuttings and 94.4% to 60% for shoot tips after 28 days of storage. The high frequency of plant regrowth from alginate-coated micropropagules coupled with high viability percentage after 28 days of storage is highly encouraging for the exchange of cassava genetic resources. Such encapsulated micropropagules could be used as an alternative to synthetic seeds derived from somatic embryos.
TL;DR: This study produced four stable tetraploid clones of A. annua L. Annua from the YUT16 hairy root clone and showed major differences in growth and artemisinin production compared to the diploid clone.
Abstract: Hairy root cultures of diploid Artemisia annua L. (clone YUT16) grow rapidly and produce the antimalarial sesquiterpene artemisinin. Little is known about how polyploidy affects the growth of transformed hairy roots and the production of secondary metabolites. Using colchicine, we produced four stable tetraploid clones of A. annua L. from the YUT16 hairy root clone. Analysis showed major differences in growth and artemisinin production compared to the diploid clone. Tetraploid clones produced up to six times more artemisinin than the diploid parent. This study provides an initial step in increasing our understanding of the role of polyploidy in secondary metabolite production, especially in hairy roots.
TL;DR: The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of A. judaica.
Abstract: An in vitro propagation system for Artemisia judaica L., a traditional Egyptian medicinal plant, has been developed. De novo shoot organogenesis was induced by culturing etiolated hypocotyls and intact seedlings on medium supplemented with thidiazuron [N-phenyl-N'-(1,2,3-thidiazol-yl) urea] via callusing at the cotyledonary notch region. Up to 16 shoots formed per seedling cultured on a medium containing 1 micro mol l(-1) thidiazuron for an optimal duration of exposure of 20 days. Regenerated shoots formed roots when subcultured onto a medium containing 1 micromol l(-1) indole-3-butyric acid. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of A. judaica.
TL;DR: The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are the use of superbinary vectors and modification of the polyamine ratio in the regeneration medium.
Abstract: Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes – vir B, -C and -G. In both cases, transient β-glucuronidase (GUS) expression ranging from 35–63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios.
TL;DR: Results indicated that the foreign DNA was successfully integrated into the orchid genome and expressed transcriptionally and translationally in these orchid plants.
Abstract: The present protocol was aimed at establishing a routine transformation procedure via Agrobacterium tumefaciens for an important Oncidium orchid cultivar. An expression vector containing hptII and gusA genes driven by the cauliflower mosaic virus (CaMV) 35S promoter was successfully introduced into the Oncidium orchid genome by A. tumefaciens. Protocorm-like bodies (PLBs) derived from protocorms, were the target explants for transformation. The transformation was performed through two stages of cocultivation, the first stage occurring on antibiotic-free medium for 3 days, and the subsequent stage on medium containing 100 mg/l timentin for 1 month. Among 1,000 inoculated PLBs, 108 putatively transformed PLBs were proliferated on 5 mg/l hygromycin selection medium. A total of 28 independent transgenic orchid plants were obtained, from which six transgenic lines that were positive for β-glucuronidase were randomly selected and confirmed by Southern, northern and western blot analyses. These results indicated that the foreign DNA was successfully integrated into the orchid genome and expressed transcriptionally and translationally in these orchid plants. The present transformation method reported is suitable for improving the Oncidium orchid through genetic engineering.
TL;DR: In this article, the authors sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots.
Abstract: Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.
TL;DR: Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars.
Abstract: Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism Out of 124 polymerase chain reaction fragments that were scored, 120 (968%) were polymorphic All the plants could be discriminated and constitute a very heterogeneous group Five unidentified almond plants found in the region of Foz Coa (north Portugal) and wild almond (P webbii) from Italy and Spain were also included Four main groups of plants could be distinguished: P dulcis cultivars; one Foz Coa plant; P webbii; and P persica (outgroup) The segregating Foz Coa plant may represent a feral individual or a hybrid between P dulcis and P webbii
TL;DR: The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains.
Abstract: The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter-gus/gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4–5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T1 descendents.
TL;DR: The ability of Agrobacterium rhizogenes to induce the formation of large amounts of root tissue on transgenic tobacco plants engineered to secrete a model recombinant protein, human secreted alkaline phosphatase (SEAP), was exploited.
Abstract: Rhizosecretion of a target protein in the hydroponic medium provides an alternative manufacturing platform that simplifies the downstream purification procedure and increases protein yield. In order to increase the production rates of rhizosecreted proteins, we have exploited the ability of Agrobacterium rhizogenes to induce the formation of large amounts of root tissue on transgenic tobacco plants engineered to secrete a model recombinant protein, human secreted alkaline phosphatase (SEAP). The secretion of SEAP from hairy roots induced on the stems of transgenic tobacco plants was 5-7 times higher than that from adventitious transgenic roots.
TL;DR: A comparison of the genetic background suggests that there is a strong genetic component amongst the different cultivars determining their regeneration capacity, and the development of these regeneration systems provides a means to use almost the whole stock plant for the efficient genetic transformation of commercial strawberry varieties.
Abstract: The parameters for optimal regeneration of seven commercial strawberry cultivars were tested using a range of explants and culture conditions. Efficient levels of regeneration – those needed to carry out transformation experiments – with the cultivars Calypso, Pegasus, Bolero, Tango and Emily were achieved with leaf discs, petioles, roots and stipules. Regeneration from cv. Elsanta proved to be difficult from all explant material, although unpollinated ovaries proved to be a promising explant source, with 12% of the explants regenerating shoots. In cv. Eros, regeneration occurred only from root tissue. A comparison of the genetic background suggests that there is a strong genetic component amongst the different cultivars determining their regeneration capacity. The development of these regeneration systems provides a means to use almost the whole stock plant for the efficient genetic transformation of commercial strawberry varieties.
TL;DR: This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides and was developed in which positive selection based on the Escherichia coli phosphomannose-isomerase gene and mannose as the selective agent was used.
Abstract: A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.
TL;DR: An improved system for routinely developing transgenic plum plants (Prunus domestica L.) through the use of Agrobacterium tumefaciens is described, the production of non-transformed "escapes" has been virtually eliminated, and rates of plant establishment in the greenhouse have been dramatically improved.
Abstract: We describe here an improved system for routinely developing transgenic plum plants (Prunus domestica L.) through the use of Agrobacterium tumefaciens. The production of non-transformed "escapes" has been virtually eliminated, and rates of plant establishment in the greenhouse have been dramatically improved. The system is based on the regeneration of shoots from hypocotyls extracted from mature seed. The shoot regeneration medium is Murashige and Skoog (MS) salts and vitamins supplemented with 7.5 microM thidiazuron and 0.25 microM indole-butyric acid. Transferring the explants after co-cultivation to shoot regeneration medium containing 80 mg l(-1) of kanamycin and 300 mg l(-1) of Timentin reduced the total number of regenerated shoots without affecting the transformation rate. Transformation rates using the described system averaged 1.2% of the hypocotyl slices producing transgenic plants, with a range of 0-4.2%. The transgenic shoots rooted at a rate of 90% on half-strength MS salts and vitamins supplemented with 5 microM alpha-naphthaleneacetic acid and 0.01 microM kinetin. Plantlets were transferred to a greenhouse directly from culture tubes with a 90% average survival.
TL;DR: Brassinolide increased the weight of loblolly pine embryogenic tissue by 66% and stimulated initiation in the more recalcitrant families of lobster pine and Douglas-fir, thus compensating somewhat for genotypic differences in initiation.
Abstract: Somatic embryogenesis (SE), the most promising technology for the large-scale production of high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and the uniformity of future U.S. forests. To be successful for commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine ( Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation percentages of loblolly pine, Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco], and Norway spruce ( Picea abies L., Karst.) were improved through the use of brassinolide. Brassinosteroids, which include brassinolide, are a relatively new group of natural plant growth regulators that are found in many plant species. They have been shown to have diverse, tissue-specific, and species-specific effects, including the stimulation of cell elongation and ethylene production and increasing resistance to abiotic stress. In our media, brassinolide was effective at concentrations ranging from 0.005-0.25 micro M. Using control medium (no brassinolide) and brassinolide-supplemented (0.1 micro M) medium, we achieved improved initiation percentages in loblolly pine, Douglas-fir, Norway spruce, and rice-15.0% to 30.1%, 16.1% to 36.3%, 34.6% to 47.4%, and 10%, respectively. Brassinolide increased the weight of loblolly pine embryogenic tissue by 66% and stimulated initiation in the more recalcitrant families of loblolly pine and Douglas-fir, thus compensating somewhat for genotypic differences in initiation. Initiation percentages in loblolly pine were improved through the combination of modified 1/2-P6 salts, 50 mg/l activated carbon (AC), adjusted levels of Cu and Zn (to compensate for adsorption by AC), 1.5% maltose, 2% myo-inositol (to raise the osmotic level, partially simulating the megagametophyte environment), 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid, 0.63 mg/l 6-benzylaminopurine, 0.61 mg/l kinetin, 3.4 mg/l silver nitrate, 10 micro M cGMP, 0.1 micro M brassinolide, and 2 g/l Gelrite. Across 12 open-pollinated families of loblolly pine, initiation percentages ranged from 2.5% to 50.7%, averaging 22.5%.
TL;DR: The results demonstrate that the α-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene.
Abstract: Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid α-linolenic acid during the seed developing stage. We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation. Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting. Furthermore, α-linolenic acid content of the T1 seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T2 and T3 progenies. These results demonstrate that the α-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene.
TL;DR: Characterisation of auxin distribution in the moss Physcomitrella patens using auxin-inducible reporter gene systems revealed that the highest auxin concentrations were in dividing and ontogenetic young cells.
Abstract: The plant hormone auxin plays a major role in a variety of growth and developmental responses, even in the more ancient plants-for example, cell differentiation in mosses. Nevertheless, almost nothing is known about the distribution of auxin during moss development. To address this question, we characterised auxin distribution in the moss Physcomitrella patens using auxin-inducible reporter gene systems. Stable transgenic Physcomitrella plants were produced expressing the beta-glucuronidase (GUS) gene driven by the auxin-inducible promoters GH3 and DR5, respectively. Both fusions showed remarkable differences with respect to auxin-induced promoter strength and expression kinetics. A detailed characterisation of the GUS expression pattern in different developmental stages revealed that the highest auxin concentrations were in dividing and ontogenetic young cells.
TL;DR: Application of this micropropagation protocol for Saussurea obvallata (DC.) Edgew has the potential to substantially reduce the pressure on natural populations.
Abstract: This is the first report of a micropropagation protocol for Saussurea obvallata (DC.) Edgew. (Asteraceae), a rare, threatened and near-endemic medicinal herb of the Indian Himalayan region. Multiple shoots were formed from epicotyle explants on Murashige and Skoog (MS) medium supplemented with 1.0 µM kinetin and 0.25 µM α-naphthaleneacetic acid. A maximum of five shoots were obtained from one explant in a 75-day culture period. The effect of subsequent subcultures on shoot formation was also studied. After 100% in vitro rooting was obtained in half-strength MS supplemented with 2.5 µM Indole-3-butyric acid, the plantlets were transferred to ex vitro conditions. Following a 15-day in vitro rooting period and 12 days of ex vitro acclimatization, 66.7% of the plantlets had established in the field. Application of this protocol has the potential to substantially reduce the pressure on natural populations
TL;DR: Microprojectile bombardment was used to identify late-pathway anthocyanin mutations, which may be responsible for the pale anthochenin coloration of important cultivars in many species but which can be difficult to characterize by other means.
Abstract: Particle bombardment was used to elucidate the function of Flavonoid3, a late-acting anthocyanin gene of the ornamental plant, carnation (Dianthus caryophyllus L.). The fl3 mutation conditions dilute anthocyanin coloration that closely resembles phenotypes produced by the anthocyanin mutants bz2 of maize and an9 of petunia. Bz2 and An9 encode glutathione S-transferases (GSTs) involved in vacuolar sequestration of anthocyanins. Constructs containing either of these or another late-function maize gene, Bronze1 (UDPglucose:flavonol 3-O-glucosyltransferase), were introduced via microprojectile bombardment into fl3 petals. Complementation resulted only from Bz2 and An9, indicating that Fl3 encodes a GST involved in the transport of anthocyanins to the vacuole. The observed result in carnation, an angiosperm phylogenetically distant from maize and petunia, indicates that GST activity might be a universal step in the anthocyanin pathway. Microprojectile bombardment was used to identify late-pathway anthocyanin mutations, which may be responsible for the pale anthocyanin coloration of important cultivars in many species but which can be difficult to characterize by other means.
TL;DR: The micropropagation method produced 6,000–8,000 shoots from two initial shoots in less than 6 months and the clonal fidelity of propagated plants was tested in Costa Rican and Indonesian pineapple farms.
Abstract: We have developed an efficient and cost-effective method for commercial micropropagation of Smooth Cayenne pineapple. In vitro shoots were used as starting materials, and either longitudinal sections of the shoots or leaf bases were used as the explants to regenerate shoots. When these explants were used, the axillary meristems, which usually remain quiescent during shoot multiplication, were able to form new shoots. Subsequent to the regeneration step, additional multiplication was achieved inside a 10-l Nalgene vessel with shoots immersed in liquid medium for 5–10 min/h (periodic immersion bioreactor, PIB). The shoots were then induced to form roots and transferred to soil. Using the above micropropagation method and the PIB, we produced 6,000–8,000 shoots from two initial shoots in less than 6 months. The clonal fidelity of propagated plants was tested in Costa Rican and Indonesian pineapple farms.