Students followed a line of reasoning and performed all the procedures even if pre
prepared material was already available for experiments and subsequent analysis. Tutors provided grown tobacco and Arabidopsis plants as well as already transformed E. coli and Agrobacteria cultures with the appropriate constructs which were also previously obtained in the laboratory and maintained in E. coli. All bacterial media were prepared by the students thus being introduced to proper aseptic procedures. Students performed plasmid preparation, bacterial transformation, protoplast preparation, rapid transient and stable transformation techniques and evaluated subcellular localization of fusion protein fluorescence in living tissues by fluorescence microscopy. During the course, students were immersed in laboratory work, while discussing important concepts pertaining genetic transformation as a molecular and biotechnological tool, cell biology and structure, molecular genetics and biotechnology.
Considering that constructs were available in the laboratory and also bacterial clones, and stably transformed Arabidopsis lines, this course provided simple and inexpensive procedures to teach genetic transformation using plants as model systems. Students contacted with laboratory equipment of the university not generally available in schools, such as electroporator, electrophoresis apparatus and fluorescence microscope.The experiments provided colorful images and conclusive results with of plant genetic transformation.
Students were introduced to the concept of transformation (introduction of foreign DNA into cells) and discussed reasons to transform cells (to introduce new genes and therefore new traits into a cell/organism).
Different techniques are used to transform different types of cells and in any type of cell, plasma membranes and/or cell walls must be penetrated without damaging the cell. Some types of plants can be transformed via infection with Agrobacterium
tumefaciens, a natural tool for plant
transformation.
Plant transformation can be transient where there is no incorporation of exogenous DNA into the genome or stable with the incorporation of introduced exogenous DNA into the genome. Steps in plant transformation include, propagate binary vector in E. coli, isolate engineered binary vector and introduce into Agrobacterium already containing a modified Ti plasmid and infect plant tissue with engineered Agrobacterium
Transient expression assays in plants can be performed by a procedure called agro- infiltration with Agrobacterium tumefaciens that transfers to the plant the T-DNA carrying the sequences in study, which will be expressed in the plant tissue. The technique consists in the infiltration of the abaxial surface of a leaf with an Agrobacterium suspension using a needle- less syringe. Expression of fluorescent fusion proteins in living plant cells was assayed by fluorescence microscopy.
In this work, we used ÀXRUHVFHQW RUJDQHOOH markers speci¿F VXEFHOOXODU FRPSDUWPHQWV the endoplasmic reticulum (ER) and the Golgi apparatus (GA), which provided an insight in the organization of the plant secretory pathway and allowed to identify subcellular distribution in living cell. Plants were transformed with a SP-GFP-HDEL construct containing the jellyfish gene encoding for the green fluorescent protein with the ER retention signal, the HDEL
sequence at the C terminus and with the SP- ST-GFP construct that encodes Sialyl- transferase, a Golgi-membrane protein, fused to GFP.The transformed cells were inspected with a fluorescent microscope. Fluorescent images of SP-GFP-HDEL showed that GFP was localized in the ER while the images of SP-ST-GFP showed that GFP localized to Golgi bodies that appeared as punctate structures (Fig. 6).
During the course, in planta transformation of Arabidopsis was also performed by dip flowering plants into Agrobacterium suspension (Fig. 2). By this simple method transformation of female gametes occurs and subsequently seeds are harvested and transformants selected. Finally, students also performed preparation of protoplasts from Arabidopsis plants (Fig. 7). Protoplasts are plant cells that have had their cell walls enzymatically removed and have been adapted a basic and versatile tool for genetic engineering and biochemical research to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and transient gene expression.
The program “Universidade Junior”, an initiative of the University of Porto, has brought to the University thousands of school students from various grades. This course configured a research-based “Summer project” targeted for secondary, pre-university students.
Students developed a project in a research laboratory, which provided opportunities to enhance their scientific literacy and envisage future prospective careers in science. Science in society issues were also approached, namely the possible social and environmental consequences of science advancements and the need for citizens to
make informed decisions regarding its applications.
Figure 6. Fluorescent images of Tobacco leaf epidermal cells transformed with GFP-HDEL
Figure 7. Arabidopsis protoplasts 8. References
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