Transient gene expression in electroporated bean cotyledon protoplasts

Bustos, Mauricio M.; Battraw, Michael J.; Kalkan, Fatma A.; Hall, Timothy C.

doi:10.1007/bf02672008

Cited by 13 publications

(3 citation statements)

References 26 publications

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“…The model presented in Figure 5 not only fits our present and previous (Bustos et a/., 1991a) observations, but also illustrates potential problems with trans-acting factors and cis-elements identified by gel retardation and in vitro footprinting assays. For example, DNA binding activities identified via in vitro approaches may not occur in a nuclear environment.…”

Section: A Proposed Model For Regulation Of Phas Transcriptionsupporting

confidence: 83%

“…Figure 4 shows the effects of the bombardment of Expression of gus driven by phas promoter sequences was also seen when chimeric constructs were supplied as naked DNA to protoplasts prepared from vegetative tissues. For example, fluorometric (MUG) assays of GUS activity in bean hypocotyl protoplasts electroporated with p795 yielded almost half the level of activity obtained using pBI221 (Bustos et al, 1991a). In addition, bean leaf proto o plasts electroporated with p795 gave approximately 50% and p295 25% of the activity obtained with pBI221 (data not shown).…”

Section: Transient Expression Ofgus From Phas/gus Constructs In Vegetmentioning

confidence: 95%

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Chromosomal integration is required for spatial regulation of expression from the β‐phaseolin promoter

et al. 1995

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SummaryThe stringency of spatial expression of phaseolin, the major storage protein of bean (Phaseolus vulgaris) seeds, has been rigorously evaluated using stable and transient transformation techniques. Transgenic tobacco plants known to be homozygous for the I~-glucuronidese (gus) reporter sequence under the regulation of various lengths of the ~-phaseolin gone (phas) promoter were shown to express gus only in developing seed tissues. No expression was detected in calli initiated from stems, leaves and immature seeds, showing that expression was not leaky in undifferentiated tissues. Control plants and cultures containing gus fused to the CaMV 35S promoter actively expressed gus under identical conditions. It was not possible to induce expression in phas/gus calli with ABA, GA or jasmonic acid. Treatment of the cultures wIth 5-azacytidine did not result in expression, excluding mathylation as the major factor regulating the phas promoter. However, strong gus expression was detected in seed of plants regenerated from these callus cultures, confirming that neIther gone rearrangements nor deletion were responsible for the lack of activity seen in tissues other than the developing seed. In contrast to the above observations, strong transient expression of guswas detected in tobacco, bean and soybean leaves following introduction of the phas/gus fusion constructs via biolistic approaches and in electroporated bean leaf and hypocotyl protoplasts. These experiments show unequivocally that the phas promoter is under rigorous spatial control when integrated into the genome, but lacks spatial control when present as extrachromosomal naked DNA. A putative model explaining these differences is presented.

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Section: A Proposed Model For Regulation Of Phas Transcriptionsupporting

confidence: 83%

Section: Transient Expression Ofgus From Phas/gus Constructs In Vegetmentioning

confidence: 95%

Chromosomal integration is required for spatial regulation of expression from the β‐phaseolin promoter

et al. 1995

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“…Immature seeds of Phaseolus vulgaris L. were obtained from greenhouse-grown plants. Protoplast preparation and transformation were performed essentially as dascdbed by Bustos et aL (1991b), with the following modifications. Bleach treatment was omitted and chopped cotyledon tissue was digested for 2-2.5 h with 1% cellulase RS and 0.1% pectolyase Y-23 at room temperature (25°C).…”

Section: Protoplast Transformationmentioning

confidence: 99%

Synergism between CACGTG (G‐box) and CACCTG cis‐elements is required for activation of the bean seed storage protein β‐phaseolin} gene^†

Kawagoe

Campell²,

Murai³

1994

The Plant Journal

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SummaryExpression of bean seed storage protein phaeeolin is under strict developmental control. Four distinct nuclear proteins recognize in vitro the proximal ,8-phaseolin promoter (-295/+45) which confers spatial and temporal regulation of the native gene. Functional significance of these protein-binding sitas was evaluated by substitution mutation of the motifs in the promoter, which was fused to GUS reporter gene, and subsequent transient gene expression assay using protopleets from developing bean cotyledons. DNA-binding protein CAN binds three CANNTG motifs, CACGTG (-248/-243), CACCTG (-163/-158), and CATATG (-100/-95). Subetifution mutation of the CACGTG motif, which is commonly known as G-box, reduced the -295 promoter activity by 75%, indicaring that the G-box is a major positive c/s-element. Mutation analyses also demonstrated that the CACCTG and CATATG motifs act as positive and negative ciselements, respectively. Substitution mutation of all three CANNTG motifs essentially eliminated the -295 promoter activity. A construct containing the G-box and CACCTG motif resulted in a transcriptional level that is much greeter than the sum of the transcriptional levels from the individual ciHlements, demonstrating that the G-box and CACCTG act synergistically. Substitution mutations of two AT-rich sequences, to which a nuclear protein AG-1 binds, showed that these sitas function as major negative (-376/-367, -356/-347) or positive (-191/-182) ciselements, and that the effect of the two AG-1 binding sites was counteractive in the -391 promoter. These

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Common Bean

Aragão

Faria²,

Peloso³

et al. 2008

Compendium of Transgenic Crop Plants

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Common bean ( Phaseolus vulgaris ) is an important source of protein in the diet of more than 300 million people throughout the world. Studies on the domestication and organization processes of the common bean genetic variability was first based on morphological traits and biochemical markers and over the last years, several research groups have made efforts in the development of microsatellite markers for common bean, those are considered the most informative class of markers. Early efforts to transform common beans demonstrated its susceptibility to Agrobacterium, and some transgenic tissues or organs, such as calli, leaves, meristems, cotyledon, and hypocotyl have been achieved. In 1993, it was the first reported P. vulgaris transformation, presenting molecular evidences of transgenic progeny. Recently, reproducible systems, based on Agrobacterium and biolistics process, have been developed to achieve transgenic bean plants routinely. Using these systems, some agronomically important genes have been introduced into common bean, such as: the methionine‐rich 2S albumin gene from Brazil nut, bar gene that confers resistance to glufosinate ammonium, LEA protein gene, and genes for resistance to the Bean golden mosaic virus (BGMV). Herbicide and BGMV‐resistant plants were the first transgenic common bean plants to be evaluated under field conditions.

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Transient gene expression in electroporated bean cotyledon protoplasts

Cited by 13 publications

References 26 publications

Chromosomal integration is required for spatial regulation of expression from the β‐phaseolin promoter

Chromosomal integration is required for spatial regulation of expression from the β‐phaseolin promoter

Synergism between CACGTG (G‐box) and CACCTG cis‐elements is required for activation of the bean seed storage protein β‐phaseolin} gene^†

Common Bean

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Transient gene expression in electroporated bean cotyledon protoplasts

Cited by 13 publications

References 26 publications

Chromosomal integration is required for spatial regulation of expression from the β‐phaseolin promoter

Chromosomal integration is required for spatial regulation of expression from the β‐phaseolin promoter

Synergism between CACGTG (G‐box) and CACCTG cis‐elements is required for activation of the bean seed storage protein β‐phaseolin} gene†

Common Bean

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Synergism between CACGTG (G‐box) and CACCTG cis‐elements is required for activation of the bean seed storage protein β‐phaseolin} gene^†