Production of transgenic maize from bombarded type II callus: Effect of gold particle size and callus morphology on transformation efficiency

Frame, Bronwyn; Zhang, Hongyi; Cocciolone, Suzy M.; Сидоренко, Л. В.; Dietrich, Charles R.; Pegg, Sue Ellen; Zhen, Shifu; Schnable, Patrick S.; Wang, Kan

doi:10.1007/s11627-000-0007-5

Cited by 162 publications

(136 citation statements)

References 31 publications

(40 reference statements)

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“…We used a particle bombardment protocol (Frame et al 2000) in which a plasmid containing a selectable marker, bar, is co-bombarded with a plasmid containing the wheat glutenin gene. The glutenin expression plasmid contains a wheat genomic DNA fragment including the 1Dx5 coding sequence and flanking sequences (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Embryogenic callus derived from the genotype Hi-II was used as a substrate for bombardment. Herbicide resistance and presence of the Glu-1Dx5 gene were used as the criteria to select callus for regeneration to T 0 plants (Frame et al 2000). The T 0 plants from 12 independent events were crossed with B73 to produce 54 ears containing F 1 kernels.…”

Section: Resultsmentioning

confidence: 99%

“…The first plasmid, pHMW1Dx5, is derived from pUC9 and contains an 8.7-kb EcoRI genomic DNA fragment from hexaploid bread wheat that includes the Glu-1Dx5 coding sequence (Anderson et al 1989), 3.2 kb of 5′ flanking sequence and 1.2 kb of 3′ flanking sequence . The construct pBAR184 consists of the maize ubiquitin promoter, first exon and first intron, which drives the Streptomyces hygroscopicus bar gene with the Agrobacterium tumefaciens nos terminator (Frame et al 2000).…”

Section: Plasmidsmentioning

confidence: 99%

“…Plant transformation was carried out at the Iowa State University Plant Transformation Facility using their standard method (Frame et al 2000). Briefly, embryogenic callus from the genotype Hi-II was co-bombarded with the plasmids pBAR184 and pHMW1Dx5.…”

Section: Plant Transformationmentioning

confidence: 99%

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Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

et al. 2002

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We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the high-molecularweight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves or embryos of plants grown from seeds expressing this protein in endosperm. Genomic Southern-blot analysis is consistent with results from SDS-PAGE and indicates that the transgene integration sites are complex and are different in the four events studied. Using the presence of this protein as a phenotypic marker, we studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and self-pollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 subunit. These data, together with PCR analysis for the transgene, suggest that the transgene is inefficiently transmitted through pollen in all four events. Abstract We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the highmolecular-weight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves or embryos of plants grown from seeds expressing this protein in endosperm. Genomic Southern-blot analysis is consistent with results from SDS-PAGE and indicates that the transgene integration sites are complex and are different in the four events studied. Using the presence of this protein as a phenotypic marker, we studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and selfpollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 subunit. These data, together with PCR analysis for the transgene, suggest that the transgene is inefficiently transmitted through pollen in all four events.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Section: Plant Transformationmentioning

confidence: 99%

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Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

et al. 2002

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“…The promoter sequence in P2P46 construct was from c-zein (black). The coding sequence (CDS, upward slanting cross hatches) and 3¢ untranslated region (UTS, wavy lines) were unchanged callus of the genotype HiII using microprojectile bombardment (Frame et al 2000). Herbicideresistant callus lines carrying the transgene were identified by PCR and were regenerated to plants.…”

Section: Development Of Transgenic Plants Expressing 1dx5 Using the Mmentioning

confidence: 99%

A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability

et al. 2007

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A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa c-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self-and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production.

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One vector‐based method to verify predicted plant miRNAs, target sequences, and function modes

Zhou

Hussain

Shi

2021

Biotech & Bioengineering

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Many microRNAs (miRNAs) have been predicted from small RNA sequencing data, but little was experimentally verified due to the lack of effective methods. Here, we developed a simple and reliable dual gene expression cassette vector-based method to verify predicted plant miRNAs. We cloned osa-miR528 as a known miRNA, hvu-miRX as a predicted miRNA and TaDREB3 open reading frame as a non-miRNA into the first gene expression cassette and fused their complementary or noncomplementary sequences as predicted target or nontarget sequences with the 3ʹ untranslated region of green fluorescent protein (GFP) in the second one. When these constructs were bombarded into plant cells, only the construct containing both osa-miR528 or hvu-miRX and its complementary sequence did not generate green fluorescence. Stem-loop reverse-transcription polymerase chain reaction detected mature osa-miR528 or mature hvu-miRX in the cells, while northern analysis showed that GFP messenger RNA from the construct containing both osa-miR528 or hvu-miRX and its complementary sequence was degraded. Taken together, the results indicate that hvu-miRX is an authentic miRNA like osa-miR528, miRNA's complementary sequence is its target sequence, and both osa-miR528 and hvu-miRX silenced the GFP expression via a cleavage mode. Our method thus facilitates the verification of predicted plant miRNAs, target sequences, and function modes. K E Y W O R D S bombardment, cleavage mode, dual gene expression cassette vector, fluorescence, green fluorescent protein, miRNA, target sequence 1 | INTRODUCTION MicroRNAs (miRNAs) are endogenous and single-stranded small RNAs (sRNAs) with the size of 20-24 nucleotides (nt). In animals, they are generated from hairpin precursors (pre-miRNAs) in the cytoplasm by Dicer, a class 3 ribonuclease III enzyme. Pre-miRNAs are generated from primary transcripts (pri-miRNAs) in the nucleus by Drosha, a class 2 ribonuclease III enzyme. In plants, both miRNAs and their hairpin pre-miRNAs are generated in the nucleus by one Dicer homolog, called Dicer-like 1 (DCL1) (Jones-Rhoades et al., 2006). Either animal pri-miRNAs or plant pri-miRNAs are most transcribed from genomic DNA by RNA polymerase II (Y. Lee et al., 2004). Only a small group associated with Alu repeats are transcribed by RNA polymerase III (Borchert et al., 2006). Plant miRNA genes are usually present as individual entities scattered around the intergenic regions. Apart from the above dominant canonical biogenesis pathway, noncanonical pathways of miRNA biogenesis such as Drosha-independent and Dicer independent pathways were also present. An example is mirtrons that are produced from the introns of mRNA during splicing (Ruby et al., 2007). Mirtron biogenesis has not been extensively documented in plants.So far only 5 and 18 putative mirtrons were found in Arabidopsis and rice, respectively (Meng & Shao, 2012). miRNAs function as a gene regulator via an RNA-induced silencing complex (RISC), which contains an argonaute (AGO) protein and other associated proteins.Overall, one m...

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Production of transgenic maize from bombarded type II callus: Effect of gold particle size and callus morphology on transformation efficiency

Cited by 162 publications

References 31 publications

Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability

One vector‐based method to verify predicted plant miRNAs, target sequences, and function modes

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