Development of an Efficient Transient Gene Expression Assay Based on Tobacco ((Nicotiana tabacum var. Xanthi) Male Gametophytes

Optimization of direct DNA delivery into tobacco ((Nicotiana tabacum var. Xanthi) male gametophytes was devised together with development of an efficient transient expression system to study gene expression under controlled conditions. Use of a GFP gene driven by strong promoter and enhancer sequences allowed an efficient non-lethal transient gene expression assay with an overall transient gene expression frequency of > 4% for uninucleate microspores and between 10 and 20% for binucleate pollen. The technique demonstrated its suitability for analysis of developmental stage-specific gene expression. The assay allowed observation of real-time transgene expression during microspore maturation proving useful for in vitro pollen selection. We have also used this protocol to determine the recombination potential of tobacco male gametic cells by assessing the frequency of extra-chromosomal homologous recombination events after co-delivery of two loss-of-function GFP genes. No increase of extrachromosomal recombination was observed in assays for transient transformation. Key words: Biolistic, GFP, Microspore, Tobacco, Nicotiana tabacum, Transformation D.O.I. 10.3329/ptcb.v19i1.4078 Plant Tissue Cult. & Biotech. 19(1): 9-23, 2009 (June)

[1]  T. Dresselhaus,et al.  Erratum to: In vitro pollen germination and transient transformation ofZea mays and other plant species , 2003, Plant Molecular Biology Reporter.

[2]  D. Honys,et al.  Expression of β-galactosidase and β-xylosidase genes during microspore and pollen development , 2005, Planta.

[3]  D. Weigel,et al.  The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene , 2005, Planta.

[4]  J. de Dios Alché,et al.  Ole e 1, the major allergen from olive (Olea europaea L.) pollen, increases its expression and is released to the culture medium during in vitro germination. , 2004, Plant & cell physiology.

[5]  J. Snape,et al.  The effect of DNA/gold particle preparation technique, and particle bombardment device, on the transformation of barley (Hordeum vulgare) , 2000, Euphytica.

[6]  D. A. Hamilton,et al.  A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements , 1998, Plant Molecular Biology.

[7]  D. Twell,et al.  Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements , 1998, Plant Molecular Biology.

[8]  V. Žárský,et al.  Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination , 1991, Sexual Plant Reproduction.

[9]  E. Heberle‐Bors,et al.  Seed set after pollination with in-vitro-matured, isolated pollen of Triticum aestivum , 1991, Theoretical and Applied Genetics.

[10]  E. Heberle‐Bors,et al.  In-situ seed production after pollination with in-vitro-matured, isolated pollen , 1988, Planta.

[11]  M. Kyo,et al.  Control of the developmental pathway of tobacco pollen in vitro , 1986, Planta.

[12]  Naveed Aziz,et al.  Efficient male germ line transformation for transgenic tobacco production without selection , 2004, Plant Molecular Biology.

[13]  Q. Yao,et al.  An improved in vitro technique for isolated microspore culture of barley , 2004, Euphytica.

[14]  D. A. Hamilton,et al.  Dissection of a pollen-specific promoter from maize by transient transformation assays , 2004, Plant Molecular Biology.

[15]  J. Feijó,et al.  Transcriptional Profiling of Arabidopsis Tissues Reveals the Unique Characteristics of the Pollen Transcriptome1[w] , 2003, Plant Physiology.

[16]  R. Mannucci,et al.  Jellyfish green fluorescent protein as a useful reporter for transient expression and stable transformation in Medicago sativa L. , 2003, Plant Cell Reports.

[17]  D. Honys,et al.  Comparative Analysis of the Arabidopsis Pollen Transcriptome1[w] , 2003, Plant Physiology.

[18]  Kazuo Tsugane,et al.  Efficient gene targeting by homologous recombination in rice , 2002, Nature Biotechnology.

[19]  E. Heberle‐Bors,et al.  Antirrhinum majus microspore maturation and transient transformation in vitro. , 2002, Journal of experimental botany.

[20]  S. Volrath,et al.  Gene targeting in Arabidopsis. , 2002, The Plant journal : for cell and molecular biology.

[21]  D. Schaefer.  Gene targeting in Physcomitrella patens. , 2001, Current opinion in plant biology.

[22]  J. Brown,et al.  Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants. , 2000, RNA.

[23]  L. Nehlin,et al.  Transient β-gus and gfp Gene Expression and Viability Analysis of Microprojectile Bombarded Microspores of Brassica napus L. , 2000 .

[24]  E. Heberle‐Bors,et al.  Microspore embryogenesis and in vitro pollen maturation in tobacco. , 1999, Methods in molecular biology.

[25]  D. Schaefer,et al.  Efficient gene targeting in the moss Physcomitrella patens. , 1997, The Plant journal : for cell and molecular biology.

[26]  E. Heberle‐Bors,et al.  Plant male germ line transformation , 1997 .

[27]  D. Prasher,et al.  Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Jim Haseloff,et al.  Mutations that suppress the thermosensitivity of green fluorescent protein , 1996, Current Biology.

[29]  J. Brown,et al.  Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency. , 1996, The Plant journal : for cell and molecular biology.

[30]  T. M. Horiagon,et al.  Deletion mapping of the Aequorea victoria green fluorescent protein. , 1996, Gene.

[31]  C. Curie,et al.  Pollen specificity elements reside in 30 bp of the proximal promoters of two pollen-expressed genes. , 1995, The Plant cell.

[32]  E. Southgate,et al.  Factors affecting the genetic engineering of plants by microprojectile bombardment. , 1995, Biotechnology advances.

[33]  D. Twell.  Molecular and Cellular Aspects of Plant Reproduction: The diversity and regulation of gene expression in the pathway of male gametophyte development , 1994 .

[34]  J C Sanford,et al.  Optimizing the biolistic process for different biological applications. , 1993, Methods in enzymology.

[35]  M. D. de Groot,et al.  Mechanisms of intermolecular homologous recombination in plants as studied with single- and double-stranded DNA molecules. , 1992, Nucleic acids research.

[36]  J. Oard.  Physical methods for the transformation of plant cells. , 1991, Biotechnology advances.

[37]  R. Offringa,et al.  Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. , 1990, The EMBO journal.

[38]  P. Benfey,et al.  Tissue‐specific expression from CaMV 35S enhancer subdomains in early stages of plant development. , 1990, The EMBO journal.

[39]  P. Benfey,et al.  Combinatorial and synergistic properties of CaMV 35S enhancer subdomains. , 1990, The EMBO journal.

[40]  J. Carrington,et al.  Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region , 1990, Journal of virology.

[41]  I. Potrykus,et al.  Intermolecular homologous recombination in plants , 1990, Molecular and cellular biology.

[42]  J. P. Mascarenhas.  Gene Activity During Pollen Development , 1990 .

[43]  M. Fromm,et al.  Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. , 1989, Plant physiology.

[44]  C. Dumas,et al.  Rapid assessment of microspore and pollen development stage in wheat and maize using DAPI and membrane permeabilization. , 1987, Stain Technology.