The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been extensively used as a marker of gene expression and protein targeting in intact cells and organisms. Since GFP has no toxic effect on cells and it is easy to detect, it can replace controversial transformation markers such as those for antibiotic or herbicide resistance. The protein can also be used to detect dynamic responses of signal transduction pathways, subcellular localisation of chimeric proteins, and transfection efficiency. It allows visualisation of the organelles and observation of many important cellular processes.
Flow cytometry (FCM) is a rapid and exact method for estimating the nuclear DNA content. Thus, it can be used for ploidy screening of different plant materials cultured in vitro (plantlets, callus, cell suspensions and somatic embryos) as well as haploids and somatic hybrids. In addition, it can be applied as a tool to analyse the events of genetic transformation. The application of FCM in biotechnology will be discussed.
In plant tissue cultures, somaclonal variation is often observed. It can be an effect of the changes in the individual chromosome number or in the ploidy level. Flow cytometry, a fast and accurate method for the estimation of the nuclear DNA content, can be applied to study these changes. The DNA content in differentiated tissues of Nicotiana tabacum cultured in vitro was estimated using Partec CCA flow cytometer, starting from explant, through callus, up to regenerated shoots. The explant constituted stem segments of N. tabacum plants, non-transformed and transformed with gfp gene. Flow cytometric analysis showed differences in the proportion of 2C, 4C, 8C and 16C cells in plant tissue in different culture stages. Among the regenerated plantlets originated from non-transformed and transformed plants, diploid, tetraploid and mixoploid forms were observed. The transformation did not influence the share of cells representing different ploidy levels in the investigated plant material.
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