Plant transformation

Plant transformation

Plant transformation is method of new gene transfer and the expression thereof, dedicated to analysis of gene functions and improvement of the characters of economically significant plants. This database discusses the main methods of plant transformation and the contribution of Lithuanian scientists into the creation, development and adaptation of these methods is highlighted.

Transformation is based on the fact that the expression of the chosen gene included into the vegetable cell is determined by the transcription and transmittance mechanisms functioning within the cell itself. There are two main types of transformation: for receiving temporary expression of genes and stable expression of genes. Plant transformation, when seeking to express the chosen gene only for certain time, is a simpler and less time consuming method. DNA is included into single plant or in vitro advocating culture cells and the expression of the transmitted gene/ genes is analysed, inter-cell localisation, induction of the promoter or the interaction of expressed proteins. Stable plant transformation is a much longer lasting process. The purpose of transformation of this type is to include DNA into cells and afterwards to regenerate them in this way receiving transformed transgene plants. Quite often stable transformation of the plants is difficult to achieve or completely impossible, even though stable transformation methods have already been adapted for most of the nutritonial, especially grain plants, as well as for plants important in the agriculture or industrial plants. Even though lately the creation of transgene plants and the growth thereof faces ethical problems, nevertheless, transformation of plants is important not only in biotechnology, but it is also one of the most important tools solving the problems of fundamental plant science.

The main Lithuanian scientific institutions, in the laboratories of which plant transformation methods are created and developed:

Institute of Biotechnology

Institute of Botany

Lithuanian Forest Research Institute

Lithuanian Institute of Horticulture

Lithuanian University of Agriculture

The Laboratory of Molecular Genetics and Biotechnology of Lithuanian Forest Research Institute (head doc. dr. Sigutė Kusienė) specializes in the biotechnology of ligneous plants http://www.mi.lt/skyriai/laboratorija.htm. The scientists of the laboratory have adapted the genetic transformation method of aspen (Populus tremula) and performed the initial investigations of the transformation of European ash (Fraxinus excelsior). This laboratory has also prepared and developed micro-reproduction of several tree species, for example of pendunculate oak (Quercus robur) and larch (Larix sp.)

Department of Genetics and Biotechnology of Orchard Plants of Lithuanian Institute of Agriculture (head prof. habil. dr. Vidmantas Stanys) http://www.lsdi.lt/genetikos_sk.htm specialises in transformation of garden and orchard plants and have successfully performed transformations of tomatoes (Lycopersicon esculentum), cherries (Prunus cerasus), quince (Cydonia oblonga) and currants (Ribes nigrum) using adventive shoots. Lithuanian Institute of Horticulture (LSDI) has developed micro-reproduction methods of most garden and orchard plants. That is a good basis for plant transformation, because namely in vitro regeneration is one of the crucial stages of transformation, and various types of the plants or even species are characterised by different regeneration characters.

Laboratory of Eukaryote Gene Engineering of Institute of Biotechnology (head dr. Gintautas Žvirblis) http://www.ibt.lt/lithuanian/labor/l4.htm and Laboratory of Agrobiotechnology of Lithuanian University of Agriculture (head doc. dr. Natalija Burbulis) http://beta.lzuu.lt/af/lt/13732&zyme=agrobiotechnologijos are developing rape (Brassica napus) transformation method: using co-cultivation with A. tumefaciens method hypocotyle explants of plants are transformed. Moreover, Institute of Biotechnology adapts transformation of thale cress (Arabidopsis thaliana) using the method of plant blossom immersion.

Laboratory of Cell Engineering of Institute of Botany (head dr. Regina Vyšniauskienė) http://www.botanika.lt/struktura/str_lab9.html. The main fields of research are genome stability investigations, creation of new forms of plant, applying somatic cell hybridization and transformation; insertion of wild plant species with valuable adaptive characters into the genome of cultivated plants. Implemented project – “Insertion of inheritable information of Physalis ixocarpa into the genome of cultivated potatoes”.

There are several main known methods of plant transformation:

The essence of the protoplast transformation method is the fact that the chosen DNA is inserted into the protoplast – into vegetable cell with eliminated membrane, which remains vital and can function as well as express the inserted DNA.

Protoplasts can be distinguished from non-differentiated cells of suspended culture or from certain organs of the plant, for example, leaves, by affecting the vegetable cells with enzymes breaking their membranes: cellulase and macerozyme or pectinase. The quality of the distinguished protoplasts mostly depends upon the quality of the initial vegetable raw material and from selecting optimal conditions for enzyme erosion of the membranes. The received protoplasts can be transformed in two ways: electroporation or polyethylenglycol (PEG). Protoplasts are electroporated, if their regeneration is planned and attempts are made to receive plant cell culture transformed in a stable way, and later on to regenerate the plants. Electroporation principle is the following one: electric pulses in the plasma membrane of the cells temporarily open hydrophilic pores, through which DNA can enter the cell. Electroporation method is sufficiently quick, reliable, inexpensive and non-toxic to the recipient cells.

Transformation using polyethylenglycol is performed when attempting temporarily express certain DNA. In such a way polyethylenglycol helps DNA to enter the cell, it has not been determined for sure, but it is supposed that having affected protoplasts with polyethylenglycol reversible permeabilization of plasma membrane is performed, during which DNA enters the cell . Transformation of protoplasts is a convenient system for the interaction of in vivo proteins, inter-cell localisation, gene expression, investigation of promoter induction.

Biolistic insertion of DNA into the cell. The principle of the method is the following one: the chosen DNA or RNA, attached to micro-particles of gold or wolfram, under influence of high pressure is triggered into the cells. After the micro-particle enters the nucleus, DNA detaches from it defunding and interacts with genomic DNA. This method can be applied for the (stable) transformation of both the nucleus and especially the plastids and mitochondrials. The advantage of the method is high efficiency of the transformation than using polyethylenglycol, while the main drawback is the net cost: expensive biolistic device is necessary as well as particles of gold or wolfram. Biolistic plant transformation method is very convenient, quick and simple enough, when analysing expression of certain genes or inter-cell localisation of proteins in vivo using reporter genes.

Plant transformation using bacteria of Agrobacterium genus (A. tumefaciens or A. rhizogenes) is a classic plant transformation method, successfully applied for various transformations of dicot and monocot plants, grassy and ligneous plants. The plants are infected by agrobacteria with plasmid, the gene of which restricted by T-DNA sequences is integrated into the chromosome of the plant.

To main modes of transformation can be distinguished, which employ bacteria of Agrobacterium genus: transformation with in vitro regeneration and direct transformation. Direct transformation is a faster way, but it can be applied for only few plant species. A. rhizogenes is most often used for transformation of roots, while A. tumefaciens is most often used for the transformation of superterranean organs. Transgene plants are separated using breeding markers and they are analyzed by performing segregational analysis, Southern hybridization, RT-PCR and other standard methods.

Transformation using viral systems. For analysis of temporary gene expression the plants can be transformed using viral systems . The main principle is the fact that transgenes are transmitted into the plants using vectors created on the basis of plant viruses. The plants can be inoculated with recombinant viral RNA or with cDNA using as an intermediary A. tumefaciens. The main advantages of the viral systems are decreased time (construction of a vector – gene/ protein expression) and efficiency, because having infected the plants, the viruses amplify quickly and spread in the whole plant. The systems of this type can be used for the expression of transgenes of various sizes . The main sphere of application of viral systems is the production of chosen proteins, most often significant in pharmacy industry, within the vegetable tissues.