Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and non-transgenic plants

Hodal, Lene; Bochardt, Anja; Nielsen, John; Mattsson, Ole; Okkels, Finn T.

doi:10.1016/0168-9452(92)90199-v

Cited by 60 publications

(37 citation statements)

References 16 publications

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“…/>acfermm, due to the lack of an eukaryotic RNA splicing appar-Likewise, introduction of malonyl CoA-ACP transacylase atus (Vancanneyt et al 1990). Intrinsic GUS activity has been (MCAT) gave no significant changes in the composition and reported in B. napus leaves (Hodal et al 1992) and in mature quantity of storage lipids. However, by use of the seed-specific embryos of B. rapa and B. oleracea (Hu et al 1990).…”

Section: Assessment Of Transgenic Plantsmentioning

confidence: 99%

Genetic transformation of Brassica

Poulsen

1996

Plant Breeding

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Tissue culture regeneration of plants from Brassica species can be achieved from various explant types. Their performance, however, varies with the genotype and age of the explant and is sensitive to medium supplements and Agrobacterium cocultivation. Most Brassica transformations were carried out using Agrobacterium and it was established that nopaline and agropine strains of .4. tumefaciens and A. rhizogenes, respectively, are the most efficient. Application of virulence gene induction is ambiguous. Binary vectors are mostly apphed in spite of indications that cointegrate vectors are more efficient. Cotransformation is successful with two different bacteria or with one bacterium carrying both plasmids, however, it has proved problematic to integrate the two transgenes into separate loci. Transformations with wild type rol genes develop hairy roots, from which transgenic plants can be regenerated. Vectorless transformation is feasible with microprojectile bombardment of microspore derived embryos being the most promising.Antibiotic markers providing resistance to kanamycin, hygromycin, and spectinomycin are applicable as selectable markers, while chloramphenicol is not suitable. Among herbicides, phosphinotricin gives good results while chlorsulfuron is unsuitable for in vitro selection. Major therapeutic antibiotics being successfully applied are carbenicillin, cefotaxime, amoxycillin, and ticarcillin. Standard methods for evaluating transgenic plants also have been applied to Brassica species. Some breeding objectives obtained through genetic transformation are presented.

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Section: Assessment Of Transgenic Plantsmentioning

confidence: 99%

Genetic transformation of Brassica

Poulsen

1996

Plant Breeding

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“…The reaction buffer (pH 8.0) is designed for specific elimination of endogenous β-glucuronidase activity in transgenic and non-transgenic tissues and plants [19].…”

Section: Transgene Expressionmentioning

confidence: 99%

Towards genetic engineering of maritime pine (Pinus pinaster Ait.)

Trontin¹,

Harvengt²,

Garin³

et al. 2002

Ann. For. Sci.

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-Using our improved protocols for somatic embryogenesis in Pinus pinaster, transgenic tissues and plantlets were recovered after microprojectile bombardment (biolistic) or cocultivation of embryonal-suspensor masses (ESM) with Agrobacterium tumefaciens. Transformation experiments were carried out with selectable hpt gene (hygromycin B resistance) and reporter gus gene (β-glucuronidase activity). With both methods, hygromycin was shown to be an effective selective agent of transformed cells within 4-19 weeks. The mean number of hygromycin-resistant lines expressing gus per gram ESM subjected to DNA transfer, ranged from 7.0 to 8.5 using biolistic and 0 to 67.3 during Agrobacterium experiments. Mature somatic embryos obtained from some transformed lines were converted into plantlets and grown in the greenhouse. The whole process (from transformation to plant acclimatisation) could be completed within only 12 months. The transgenic state of ESM, somatic embryos and plants was confirmed by histochemical GUS assays and molecular methods.

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“…In different species optimal endogenous GUS-like activity has been reported between pH 4 and pH 6 (Hodal et al 1992), whereas it is histochemically undetectable at pH 7.0 or higher (Alwen et al 1992;Hodal et al 1992;Sudan et al 2006;Solís-Ramos et al 2010a). In contrast, the pH of E. coli-derived GUS has optimum activity at pH 7.0 (Jefferson 1987).…”

Section: Resultsmentioning

confidence: 99%

Transient genetic transformation of embryogenic callus of Cocos nucifera

et al. 2011

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Coconut palm (Cocos nucifera) is a plant species recalcitrant to in vitro morphogenesis and no protocols for the genetic transformation of coconut tissues have been published. The present study aimed to develop a protocol for genetic transformation of this palm species; evaluating reporter genes, transformation methods, and conditions for the use of antibiotics to select transformed plant cells. The gene gusA was first used for Agrobacterium tumefaciens mediated transformation of coconut embryogenic calli. However, endogenous GUS-like activity was found in calli not co-cultured with bacteria. Then essays for Agrobacterium-mediated transformation were developed using green and red fluorescent genes. Both genes are suitable as reporter genes for coconut transformation. In order to establish a protocol for coconut genetic transformation, an approach was used that combined biobalistics to generate micro-wounds in explants, vacuum infiltration and co-culture with Agrobacterium tumefaciens (C58C1 + pER10W-35SRed containing the embryogenesis related gene WUSCHEL). Calli treated with the combined protocol showed red fluorescence with greater intensity and greater area than calli treated with either biobalistics or infiltration, followed by bacteria co-culture. PCR amplification of DNA extracts from transformed embryogenic callus produced a band with the expected size using WUSCHEL primers (862 bp). No band was obtained using the VirE2 primers. This is the first report of transient genetic transformation of C. nucifera and it is the first step toward a protocol that will be useful for the study of the role of genes of interest and for practical applications, such as the improvement of coconut micropropagation via somatic embryogenesis.

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Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and non-transgenic plants

Cited by 60 publications

References 16 publications

Genetic transformation of Brassica

Genetic transformation of Brassica

Towards genetic engineering of maritime pine (Pinus pinaster Ait.)

Transient genetic transformation of embryogenic callus of Cocos nucifera

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