Transient gene expression is a powerful tool to study gene function in plants. In citrus, transfo... more Transient gene expression is a powerful tool to study gene function in plants. In citrus, transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than . Here we describe an improved method for gene expression in epidermal cells of citrus leaves, using the Bio-Rad Helios gene-gun. Gene-gun bombardment of GFP-HDEL produced highly efficient gene expression in large number of cells and in different citrus varieties. We show here that transiently expressed proteins have maintained their functions in plants, and this is demonstrated by the subcellular localization of different organelle markers, and by a functional assay of effector AvrGF1. To further expand the availa...
Biotechnology & genetic engineering reviews, Jan 2, 2017
Development of in vitro plant regeneration method from Brassica explants via organogenesis and so... more Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.
DESCRIPTION A Mature Citrus Facility was established at the University of Florida’s Citrus Resear... more DESCRIPTION A Mature Citrus Facility was established at the University of Florida’s Citrus Research and Education Center to produce transgenic citrus tolerant to canker and greening bacterial diseases.
Citrus canker and greening are causing severe economic losses in Florida by decreasing fruit qual... more Citrus canker and greening are causing severe economic losses in Florida by decreasing fruit quality, yield, and negatively affecting tree health. Traditional breeding programs for citrus are not very helpful in the fight against these diseases because the time for flowering and fruit production from seed of new cultivars can range from 3-20 years. Mature tissue transformation technology enables genetic improvement and the early flowering and fruiting of scion. The Agrobacterium transformation protocol was adapted to scion sweet orange cultivars 'Valencia', 'Hamlin', and 'Pineapple', and 'Ray Ruby' grapefruit, which are important to the Florida citrus industry. Scion cultivars were transformed with Agrobacterium strain EHA 105 harboring binary vectors, pCambia 2301 or pTLAB21, containing the nptII selectable marker and GUS or GFP reporter genes, respectively. Transgenic plants were identified by PCR, NPTII immunostrip tests and GUS staining or GFP flu...
One of the most promising directions in the study of phenotypic plasticity is its detailed analys... more One of the most promising directions in the study of phenotypic plasticity is its detailed analysis in organisms that are also well-studied in other aspects. Also, conclusions based on plasticity studies in environmental gradients that closely mimic natural variation are shown to be the most relevant. Following those directions, we conducted this study of phenotypic plasticity on the currently best available model system in flowering plants--Arabidopsis thaliana, and utilized one of the most common variations experienced in the wild--variation in density. Four Arabidopsis thaliana commonly used inbred lines (ecotypes) were grown in densities from one to seven plants per pot. Both phenotypic plasticity and its genetic variability were detected for almost all of 11 analyzed traits, with analyzed ecotypes responding strongly to density of just two plants per pot. Density had small effect on life history and moderate effect on size traits, while vegetative and reproductive traits respon...
Etiolated seedlings of Arabidopsis thaliana nutated under conditions of physiological darkness wh... more Etiolated seedlings of Arabidopsis thaliana nutated under conditions of physiological darkness while about ten percent of monitored individuals exhibited regular elliptical nutation, circumnutation. Pre-irradiation with red light prevented occurrence of circumnutation without having an effect on the average rate of the nutational movement. Phototropic response of seedlings to unilateral blue light appeared to be superimposed over nutation. Throughout gravitropism, some seedlings continued to exhibit nutation suggesting that these two processes are independently controlled. Based on these results, we suggest that nutation in Arabidopsis probably is not controlled by the mechanism predicted by the theory of gravitropic overshoots.
Transient gene expression is a powerful tool to study gene function in plants. In citrus, transfo... more Transient gene expression is a powerful tool to study gene function in plants. In citrus, transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than . Here we describe an improved method for gene expression in epidermal cells of citrus leaves, using the Bio-Rad Helios gene-gun. Gene-gun bombardment of GFP-HDEL produced highly efficient gene expression in large number of cells and in different citrus varieties. We show here that transiently expressed proteins have maintained their functions in plants, and this is demonstrated by the subcellular localization of different organelle markers, and by a functional assay of effector AvrGF1. To further expand the availa...
Biotechnology & genetic engineering reviews, Jan 2, 2017
Development of in vitro plant regeneration method from Brassica explants via organogenesis and so... more Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.
DESCRIPTION A Mature Citrus Facility was established at the University of Florida’s Citrus Resear... more DESCRIPTION A Mature Citrus Facility was established at the University of Florida’s Citrus Research and Education Center to produce transgenic citrus tolerant to canker and greening bacterial diseases.
Citrus canker and greening are causing severe economic losses in Florida by decreasing fruit qual... more Citrus canker and greening are causing severe economic losses in Florida by decreasing fruit quality, yield, and negatively affecting tree health. Traditional breeding programs for citrus are not very helpful in the fight against these diseases because the time for flowering and fruit production from seed of new cultivars can range from 3-20 years. Mature tissue transformation technology enables genetic improvement and the early flowering and fruiting of scion. The Agrobacterium transformation protocol was adapted to scion sweet orange cultivars 'Valencia', 'Hamlin', and 'Pineapple', and 'Ray Ruby' grapefruit, which are important to the Florida citrus industry. Scion cultivars were transformed with Agrobacterium strain EHA 105 harboring binary vectors, pCambia 2301 or pTLAB21, containing the nptII selectable marker and GUS or GFP reporter genes, respectively. Transgenic plants were identified by PCR, NPTII immunostrip tests and GUS staining or GFP flu...
One of the most promising directions in the study of phenotypic plasticity is its detailed analys... more One of the most promising directions in the study of phenotypic plasticity is its detailed analysis in organisms that are also well-studied in other aspects. Also, conclusions based on plasticity studies in environmental gradients that closely mimic natural variation are shown to be the most relevant. Following those directions, we conducted this study of phenotypic plasticity on the currently best available model system in flowering plants--Arabidopsis thaliana, and utilized one of the most common variations experienced in the wild--variation in density. Four Arabidopsis thaliana commonly used inbred lines (ecotypes) were grown in densities from one to seven plants per pot. Both phenotypic plasticity and its genetic variability were detected for almost all of 11 analyzed traits, with analyzed ecotypes responding strongly to density of just two plants per pot. Density had small effect on life history and moderate effect on size traits, while vegetative and reproductive traits respon...
Etiolated seedlings of Arabidopsis thaliana nutated under conditions of physiological darkness wh... more Etiolated seedlings of Arabidopsis thaliana nutated under conditions of physiological darkness while about ten percent of monitored individuals exhibited regular elliptical nutation, circumnutation. Pre-irradiation with red light prevented occurrence of circumnutation without having an effect on the average rate of the nutational movement. Phototropic response of seedlings to unilateral blue light appeared to be superimposed over nutation. Throughout gravitropism, some seedlings continued to exhibit nutation suggesting that these two processes are independently controlled. Based on these results, we suggest that nutation in Arabidopsis probably is not controlled by the mechanism predicted by the theory of gravitropic overshoots.
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Papers by Vladimir Orbovic