Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green-fluorescent protein

Carlson, Alfred; Letarte, Jocelyne; Chen, J.; Kasha, K. J.

doi:10.1007/s002990100328

Cited by 47 publications

(26 citation statements)

References 28 publications

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“…With this pretreatment, Jähne et al (1994) reported that transgenic fertile plants were homozygous for the transgene. In contrast, Yao et al (1997) and Carlson et al (2001) produced hemizygous transgenic plants with 0.3 M mannitol at 24C for 4 days and with 0.3 M mannitol at 4C for 4 days, respectively. The reason for heterozygosity is likely related to the developmental stage of microspores at the time of bombardment or of DNA integration, rather than the stage right after pretreatment.…”

Section: Resultsmentioning

confidence: 79%

“…If microspores were at the G1 stage after bombardment, the transformation and subsequent chromosome doubling should result in homozygous transgenic plants in one step. However, in barley, a 4-day pretreatment of 0.3 M mannitol plus cold followed by biolistic bombardment procedures led to hemizygous transgenic plants (Carlson et al 2001), as did pretreatment for 4 days in 0.3 M mannitol at 24C (Yao et al 1997). Conversely, a 28-day cold pretreatment, which slowed but did not block mitotic division (Hu and Kasha 1999), led to homozygous transgenics using biolistic procedures (Jähne et al 1994).…”

Section: Introductionmentioning

confidence: 93%

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The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley (Hordeum vulgare L.) uninucleate microspores

Shim

Kasha

2003

Plant Cell Reports

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The objective of this study was to correlate the time that DNA synthesis first occurs in haploid microspores of barley with cell cycle and plant morphological stages and to subsequently assess the influence of pretreatments on DNA synthesis at different stages of microspore development. Spikes with microspores in early, mid, and late uninucleate stages of the two-rowed barley cultivars Manley and Igri were subjected to two commonly used pretreatments. First, during cold pretreatment for 28 days there was a slow increase in relative DNA values as well as asymmetric nuclear divisions in some microspores. Second, during a 4-day cold plus 0.3 M mannitol pretreatment, there was very little change in the microspore stage or DNA values indicating that for the duration of this pretreatment the progression of the cell cycle was essentially suspended at all stages, both in Igri and Manley. The results are discussed relative to the potential for genetic transformation of microspores.

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Section: Resultsmentioning

confidence: 79%

Section: Introductionmentioning

confidence: 93%

The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley (Hordeum vulgare L.) uninucleate microspores

Shim

Kasha

2003

Plant Cell Reports

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“…Since the first detection of transgenes in petunia plants Hoopen et al 1996), reports have appeared describing the use of FISH in transgenic cereal crops (Pedersen et al 1997;Abranches et al 2000;Leggett et al 2000;Svitashev et al 2000;Carlson et al 2001;Salvo-Garrido et al 2001).…”

Section: Introductionmentioning

confidence: 99%

Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.)

2002

Theor Appl Genet

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Introduced transgenes, uidA, sgfp (S65T) and/or bar, were localized using fluorescence in situ hybridization (FISH) on metaphase chromosomes of transgenic barley produced by microparticle bombardment of immature embryos. Of the 19 independent transgenic lines (eight diploid and 11 tetraploid), nine had uidA and ten had s gfp (S65T). All lines tested had three or more copies of the transgenes and 18 out of 19 lines had visibly different integration sites. At a gross level, it appeared that no preferential integration sites of foreign DNA among chromosomes were present in the lines tested; however, a distal preference for transgene integration was observed within the chromosome. In diploid T0 plants that gave a 3:1 segregation ratio of transgene expression in the T1, only single integration sites were detected on one of the homologous chromosomes. Homozygous diploid plants had doublet signals on a pair of homologous chromosomes. All tetraploid T0 plants that gave a 3:1 segregation ratio in the T1 generation had only a single integration site on one of the homologous chromosomes. In contrast, the single tetraploid T0 plant with a 35:1 segregation ratio in the T1 generation had doublet signals on a pair of homologous chromosomes. In the one tetraploid T0 line, which had a homozygote-like segregation ratio (45:0), there were doublet signals at two loci on separate chromosomes. We conclude that the application of FISH for analysis of transgenic plants is useful for the gross localization of transgene(s) and for early screening of homozygous plants.

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“…The number of GFP-expressing cells began to decrease after day 2 (Fig. 2) in all types of bombarded tissues and had declined to a very low level by 1 week post-bombardment, as was also observed by Ponappa et al (1999) in soybean and Carlson et al (2001) in barley microspores.…”

Section: Resultsmentioning

confidence: 57%

Evaluation of different promoters driving the GFP reporter gene and selected target tissues for particle bombardment of Dendrobium Sonia 17

et al. 2003

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Three different morphological callus types, identified as type A, B and C, and tips of in vitro inflorescences were used as target tissues for genetic transformation. Five different DNA plasmids carrying a synthetic green fluorescent protein (gfp) gene driven by different promoters, CaMV 35S, HBT, and Ubi1 were tested for the genetic transformation of Dendrobium Sonia 17. 35S-sgfp-TYG-nos (p35S) with the CaMV 35S promoter showed the highest GFP transient expression rate, while the HBT and Ubi1 promoters showed a relatively lower expression rate in all of the target tissues tested. The highest number of GFP-expressing cells was observed on day 2 post-bombardment, and the number declined gradually over the course of the next 2 weeks. Type A and B callus were found to be the best potential target tissues for genetic transformation.

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Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green-fluorescent protein

Cited by 47 publications

References 28 publications

The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley (Hordeum vulgare L.) uninucleate microspores

The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley (Hordeum vulgare L.) uninucleate microspores

Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.)

Evaluation of different promoters driving the GFP reporter gene and selected target tissues for particle bombardment of Dendrobium Sonia 17

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