Summary
β-glucuronidase (GUS) expression driven with different promoter constructs was quantitatively and histologically compared in peanut leaf tissue following microprojectile bombardment. X-Gluc staining patterns varied with the construct used. Tissues bombarded with the pAC2GUS construct had larger foci and a greater percentage of area staining blue. pEmuGN exhibited the greatest numbers of blue spots compared to pAC2GUS and pTRA140. Histological evaluations of blue staining foci showed a diffusion gradient of blue precipitate from a central, prominently-staining cell outward to as many as seven cell layers. The intensity of X-gluc product in centrally-staining cells varied. Gold microprojectile particles were usually located within the three surface cell layers. Depending on the plasmid construct, 72–90% of the centrally-staining cells had at least one gold particle. However, the presence of GUS expression did not appear to require a microprojectile within the nucleus, which was observed in 37% or fewer of the centrally-staining cells. With the pAC2GUS construct, staining patterns varied with location within leaflets and had an “edge effect,” i.e., blue spots were frequently larger at the margin versus central regions. This enhanced activity could be anticipated with an actin promoter in the more mitotically active marginal leaf cells. Total GUS activity as determined by fluorometric analyses was correlated with the percentage of X-gluc stained area. The pAC2GUS construct exhibited the highest total GUS activity among the three constructs.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. Molecular biology of the cell. 2nd ed. Garland Pub., New York; 1989.
Aragao, F. J. L.; Grossi de Sa, M. F.; Davey, M. R.; Brasileiro, A. C. M.; Faria, J. C.; Rech, E. L. Factors influencing transient gene expression in bean (Phaseolus vulgaris L.) using an electrical particle acceleration device. Plant Cell Rep. 12:483–490; 1993.
Caissard, J. C.; Guivarc’h, A.; Rembur, J.; Azmi, A.; Chriqui, D. Spurious localization of diX-indigo microcrystals generated by the histochemical GUS assay. Transgenic Res. 3:176–181; 1994.
Chibbar, R. N.; Kartha, K. K.; Datla, R. S. S.; Leung, N.; Caswell, K.; Mallard, C. S.; Steinhauer, L. The effect of different promoter-sequences on transient expression of gus reporter gene in cultured barley (Hordeum vulgare L.) cells. Plant Cell Rep. 12:506–509; 1993.
Chowdhury, M. K. U.; Parveez, G. K. A.; Saleh, N. M. Evaluation of five promoters for use in transformation of oil palm (Elaeis guineensis Jacq.). Plant Cell Rep. 16:277–281; 1997.
Flavell, R. B.; Dart, E.; Fuchs, R. L.; Fraley, R. T. Selectable marker genes: safe for plants? Bio/Technology 10:141–144; 1992.
Gallagher, S. R. Quantitation of GUS activity by fluorometry. In: Gallagher, S. R., ed. GUS protocols. San Diego: Academic Press; 1992:47–59.
Gallo-Meagher, M.; Irvine, J. E. Effects of tissue type and promoter strength on transient GUS expression in sugarcane following particle bombardment. Plant Cell Rep. 12:666–670; 1993.
Gamborg, O.; Miller, R.; Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell. Res. 50:150–158; 1968.
Hensgens, L. A. M.; De Bakker, E. P. H. M.; van Os-Ruygrok, E. P.; Rueb, S.; van de Mark, F.; van der Maas, H. M.; van der Veen, S.; Kooman-Gersmann, M.; Hart, L.; Schilperoort, R. A. Transient and stable expression of gusA fusions with rice genes in rice, barley and perennial ryegrass. Plant Mol. Biol. 22:1101–1127; 1993.
Hunold, R.; Bronner, R.; Hahne, G. Early events in microprojectile bombardment: cell viability and particle location. Plant J. 5:593–604; 1994.
Klein, T. M.; Gradziel, T.; Fromm, M. E.; Sanford, J. C. Factors influencing gene delivery into Zea mays cells by high-velocity microprojectiles. Bio/Technology 6:559–563; 1988.
Last, D. I.; Brettell, R. I. S.; Chamberlain, D. A.; Chaudhury, A. M.; Larkin, P. J.; Marsh, E. L.; Peacock, W. J.; Dennis, E. S. pEmu: an improved promoter for gene expression in cereal cells. Theor. Appl. Genet. 81:581–588; 1991.
McElroy, D.; Zhang, W.; Cao, J.; Wu, R. Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171; 1990.
Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.
Rathus, C.; Bower, R.; Birch, R. G. Effects of promoter, intron and enhancer elements on transient gene expression in sugarcane and carrot protoplasts. Plant Mol. Biol. 23:613–618; 1993.
Reggiardo, M. I.; Arana, J. L.; Orsaria, L. M.; Permingeat, H. R.; Spitteler, M. A.; Vallejos, R. H. Transient transformation of maize tissues by microparticle bombardment. Plant Sci. 75:237–243; 1991.
Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: a laboratory manual. Cold Springer Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
Stomp, A. M. Histochemical localization of β-glucuronidase. In: Gallagher, S. R., ed. GUS protocols. San Diego: Academic Press; 1992:103–113.
Taylor, M. G.; Vasil, V.; Vasil, I. K. Enhanced GUS gene expression in cereal/grass cell suspensions and immature embryos using maize ubiquitin-based plasmid pAHC25. Plant Cell Rep. 12:491–495; 1993.
Walter, C.; Smith, D. R.; Connett, M. B.; Grace, L.; White, D. W. R. A biolistic approach for the transfer and expression of a Gus reporter gene in embryogenic cultures of Pinus radiata. Plant Cell Rep. 14:69–74; 1994.
Yamashita, T.; Lida, A.; Morikawa, H. Evidence that more than 90 percent of β-glucuronidase-expressing cells after particle bombardment directly receive the foreign gene in their nucleus. Plant Physiol. 97:829–831; 1991.
Zheng, Z.; Hayashimoto, A.; Li, Z.; Murai, N. Hygromycin resistance gene cassettes for vector construction and selection of transformed rice protoplasts. Plant Physiol. 97:832–835; 1991.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kim, T., Chowdhury, M.K.U. & Wetzstein, H.Y. A quantitative and histological comparison of gus expression with different promoter constructs used in microprojectile bombardment of peanut leaf tissue. In Vitro Cell.Dev.Biol.-Plant 35, 51–56 (1999). https://doi.org/10.1007/s11627-999-0009-x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11627-999-0009-x