Gene transfer to plants by electroporation: methods and applications.

Developing gene transfer technologies enables the genetic manipulation of the living organisms more efficiently. The methods used for gene transfer fall into two main categories; natural and artificial transformation. The natural methods include the conjugation, transposition, bacterial transformation as well as phage and retroviral transductions, contain the physical methods whereas the artificial methods can physically alter and transfer genes from one to another organisms' cell using, for instance, biolistic transformation, micro- and macroinjection, and protoplast fusion etc. The artificial gene transformation can also be conducted through chemical methods which include calcium phosphate-mediated, polyethylene glycol-mediated, DEAE-Dextran, and liposome-mediated transfers. Electrical methods are also artificial ways to transfer genes that can be done by electroporation and electrofusion. Comparatively, among all the above-mentioned methods, electroporation is being widely used owing to its high efficiency and broader applicability. Electroporation is an electrical transformation method by which transient electropores are produced in the cell membranes. Based on the applications, process can be either reversible where electropores in membrane are resealable and cells preserve the vitality or irreversible where membrane is not able to reseal, and cell eventually dies. This problem can be minimized by developing numerical models to iteratively optimize the field homogeneity considering the cell size, shape, number, and electrode positions supplemented by real-time measurements. In modern biotechnology, numerical methods have been used in electrotransformation, electroporation-based inactivation, electroextraction, and electroporative biomass drying. Moreover, current applications of electroporation also point to some other uncovered potentials for various exploitations in future.

Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos.

We report a new protocol for the stable transformation of Larix gmelinii. Thirty mature zygotic embryos precultured for 3 days on solid medium supplemented with benzyladenine were bombarded with plasmids pUC-GHG (GUS, HPT, and GFP genes) or pBI221-HPT (HPT and GUS genes). After a 2-month culture on selection medium, hygromycin-resistant calli appeared on the surfaces of the necrotic embryos. The frequencies of embryos with resistant calli were 18.4% and 17.4% in the transformations with pUC-GHG and pBI221-HPT DNA, respectively. More than 20 adventitious shoots formed from each of the transgenic calli. Of 17 elongated shoots selected for culturing on a rooting medium, five shoots rooted after 2 months. Expression of the GFP and GUS genes was detected in the resistant tissues by microscopic observations and by a histological GUS activity assay, respectively. PCR and Southern analysis confirmed the stable insertion of the introduced DNA into the genome.

Neuronal transfection using particle-mediated gene transfer.

This unit describes the use of particle-mediated gene transfer (also known as biolistics) for the transfection of neuronal cell lines and brain slices. Like nuclear microinjection of DNA, biolistics results in the direct introduction of DNA into the nucleus; it is perhaps for this reason that biolistics works as well in mitotic cells as in postmitotic cells such as skeletal muscle, skin, liver, and neurons. The basic principle of biolistics is to accelerate micron-sized gold particles coated with DNA towards target cells or tissue. Cells penetrated by these particles have a high likelihood of being transfected by the DNA thus introduced. The motive force for particle acceleration can come from a variety of sources, the most widely used is described in this unit and is a supersonic shock wave generated by the rupture of a kapton membrane induced by high-pressure helium. Another option included in this unit is to propel the gold particles by gas jet entrainment.