Transgenic barley (Hordeum vulgare L.) by electroporation of protoplasts

Salmenkallio-Marttila, Marjatta; Aspegren, Kristian; Åkerman, Satu; Kurten, Ulrika; Mannonen, Leena; Ritala, Anneli; Teeri, Teemu H.; Kauppinen, Veli

doi:10.1007/bf00193741

Cited by 50 publications

(18 citation statements)

References 20 publications

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“…PVA cDNA (7 mg) was introduced into batches of 10 6 protoplasts by electroporation with a Bio-Rad Genepulser II as described (Denecke and Vitale, 1995) except that the electroporation buffer of Salmenkallio-Marttila et al (1995) was used. Electroporated protoplasts were incubated at room temperature under dim light for 2 to 3 days, after which they were harvested by low-speed centrifugation (2000g for 5 min at 208C).…”

Section: Preparation and Inoculation Of Protoplastsmentioning

confidence: 99%

Control of Nuclear and Nucleolar Localization of Nuclear Inclusion Protein a of Picorna-LikePotato virus AinNicotianaSpecies

2009

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The multifunctional nuclear inclusion protein a (NIa) of potyviruses (genus Potyvirus; Potyviridae) accumulates in the nucleus of virus-infected cells for unknown reasons. In this study, two regions in the viral genome-linked protein (VPg) domain of NIa in Potato virus A (PVA) were found to constitute nuclear and nucleolar localization signals (NLS) in plant cells (Nicotiana spp). Amino acid substitutions in both NLS I (residues 4 to 9) and NLS II (residues 41 to 50) prevented nuclear localization, whereas mutations in either single NLS did not. Mutations in either NLS, however, prevented nucleolar localization and prevented or diminished virus replication in protoplasts, accumulation in infected plant tissues, and/or systemic movement in plants. One NLS mutant was partially complemented by the wild-type VPg expressed in transgenic plants. Furthermore, NLS I controlled NIa accumulation in Cajal bodies. The VPg domain interacted with fibrillarin, a nucleolar protein, and depletion of fibrillarin reduced PVA accumulation. Overexpression of VPg in leaf tissues interfered with cosuppression of gene expression (i.e., RNA silencing), whereas NLS I and NLS II mutants, which exhibited reduced nuclear and nucleolar localization, showed no such activity. These results demonstrate that some of the most essential viral functions required for completion of the infection cycle are tightly linked to regulation of the NIa nuclear and nucleolar localization.

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Section: Preparation and Inoculation Of Protoplastsmentioning

confidence: 99%

Control of Nuclear and Nucleolar Localization of Nuclear Inclusion Protein a of Picorna-LikePotato virus AinNicotianaSpecies

2009

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“…Thus, numerous other methods for DNA transfer into the regenerative competent cells were developed for cereals which were also applied to barley. Amongst these methods, direct DNA transfer into protoplasts was easily achieved due to the absence of the cell wall; however, the fi rst report on successful culture and selection of transgenic barley callus lines was published only in 1991 , and it took another 4 years that fertile transgenic barley plants were generated via the protoplast approach using polyethylene glycol-mediated DNA uptake (Funatsuki et al 1995 ;Kihara et al 1998 ), followed by electroporation (Salmenkallio-Marttila et al 1995 ) and microinjection in zygote protoplasts (Holm et al 2000 ). Alternative methods were employed to circumvent diffi culties of barley cell culture like imbibing of embryos in DNA (Töpfer et al 1989 ), electrophoresis of DNA into germinating seeds (Ahokas 1989 ) and macroinjection of DNA into fl oral tillers or application of plasmid-DNA to stigmas (Mendel et al 1990 ).…”

Section: Development Of Dna Delivery Techniquesmentioning

confidence: 98%

Barley

Schulze¹

2013

Alien Gene Transfer in Crop Plants, Volume 2

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Barley ( Hordeum vulgare L.) is a widely adapted cereal crop with an extremely wide geographic distribution throughout the world. It fi nds great use for animals as a feed and for humans as a grain, especially as the source for malt for the brewing industry. In recent times, there is considerable interest in the nutritional properties of barley due to the discovery of the cholesterol-lowering effect of β-glucan, a cell wall polysaccharide. Exploitation of genetic diversity in the primary and secondary gene pool of barley using DNA-based technologies has yielded interspecifi c crosses with improved grain properties, malting quality and resistance to biotic and abiotic stresses. The signifi cant achievements regarding introgression of alien genes include the genes Rym14(Hb) , Rym16(Hb) and Ryd4(Hb) which were introgressed from Hordeum bulbosum conferring resistance to BaMMV, BaYMV and BYDV in barley. Signifi cant advances in genetic engineering of barley have been obtained, and strategies for establishment of regenerative cell and tissue culture systems as well as for development of DNA delivery techniques have been formulated. Lately, a huge potential has been realised in barley grains to produce pharmaceutical proteins like oral vaccines, growth supplements and food additives which are being exploited in a commercial scale. Nevertheless, several problems still remain like the strong genotype dependency of barley transformation protocols, transformation effi ciency, transgene stability and public acceptance. The review focuses on all these issues and elaborates achievements made in the last two decades in genetic enhancement of barley using different alien gene transfer approaches.

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“…To date, the Agrobacterium-and biolistic-mediated transformation methods remain the very successful methods in soybean transformation, whereas other available transformation technologies have not been practical in soybean, which include electroporation-mediated transformation [72], PEG/liposome-mediated transformation [73], silicon carbide-mediated transformation [74], microinjection [75] and chloroplast-mediated transformation [76]. Of these two, Agrobacterium-mediated transformation has become more adapted in public laboratories worldwide.…”

Section: New Directions Of Soybean Genetic Engineering Skills and Vementioning

confidence: 99%