The plants,in which a functional foreign gene has been incorporated by any biotechnological methods that generally not present in plant, are called transgenic plants. However, a number of transgenic plants carrying genes for traits of economic importance have either been released for commercial cultivation or are under field trials. There are several methods discussed in previous sections of biotechnology in this website which are used in gene transfer. These includes: (i)
particle bombardment, (iii)
microinjection, (iv) Agrobacterium-mediated
gene transfer, (v) co-cultivation (protoplast transformation) method, (vi)
leaf disc transformation method, (vii)
virus-mediated transformation, (viii)
pollen-mediated transformation, (ix)
liposome-mediated transformation, etc. Initially, some plants were produced by using reporter genes (see preceding section). Later on several genes for known traits of economic importance were incorporated into many crop plants. In others promoter sequences have been used that reduced/enhamced tissue specific expression of the adjacent genes according to requirement In some cases antisense RNA genes have been introduced to inhibit expression of some existing genes in a desirable manner. All these approaches led to the development of transgenic crop plants of economic importance. More than 1000 field trial tests with transgenic crop plant have been conducted. Some of the commercially grown transgenic crop plants in developed countries are: 'Flavr Savri' and 'Endless Summer' tomatoes, 'Freedom IF squash, 'High-lauric' rapeseed (canola) and 'Roundup Ready' soybean, etc. During 1995, in the USA full registration was granted to genetically engineered Bt
gene containing insect resistant 'New Leaf (potato), 'Maximized (corn), 'BollGard' (cotton) (Gupta, 1996).
So far more than 60 transgenic dicot plants including herbs, shrubs and trees and several monocots (e.g.
maize, oat, rice, wheat, etc.) have been produced. In future the number of these crops certainly will go up. These transgenic plants contain certain selected traits such as herbicide resistance, insect resistance, virus resistance, seed storage protein, modified ripening, modified seed oil, agglutinin, etc. (Table 9.5). Moreover, in the light of future need the transgenic plants are being looked up as bioreactor for molecular farming i.e.
for the production of novel biomedical drugs such as growth hormones, vaccines, antibodies, interferon, etc. (see
Table 9.5. Transgenic plants produced by different methods.
Selectable Marker Genes and Their Use in Transformed Plants
||Transgenic plant species
||Asparagus sp., Dactylis glomerata, Secale, cereale, Oryza sativa, Triticum aestivum, Zea mays, Avena sativa, Festulaca arundinacea.
||Armorcia sp., Nicotina tabaccum, N. plumbaginifolia, Petunia hybrida, Lycopersicon esculentum, Solanum tuberosum, S. melangana, Arabdiopsis thaliana, Lactuca sativa, Apium grareolens, Helianthus annus, Linum usitatisimum, Brassica napus, B. oleracea, B. oleracea var capita, B. compestris, Gosssypium hirsutum, Beta vulgaris, Glycine max, Pisum sativum, Medicago sativa, M. varia, Lotus corniculanum, Vigna aconitifolia, Cuccumis sativus, C. melo, Cichorium intybus, Daccus carrota, Glycorrhiza glabra, Digitalis purpurea, Ipomoea batata, I. purpurea, Fragaria sp., Actinidia sp., Carica papaya, Vitis vinifera, Dianthus caryophyllus, Vaccinium macrocarpon, Chrysanthemum sp., Rosa sp., Populous sp. Melus sylvestris, Pyrus communis, Azadirachta indica, Juglans regia.
When plant cells are transformed by any of the transformation methods as given earlier, it is necessary to isolate the transformed cells/tissue. However, it is possible to do now. There are certain selectable marker genes present in vectors that facilitate the selection process. In transformed cells the selectable marker genes are introduced through vector. The transformed cells are cultured on medium containing high amount of toxic level of substrates such as antibiotic, herbicides, etc. For each marker gene there is one substrate (Table 9.6). For a model transgenic system, tobacco is the most common plant that is found everywhere. The young explants such as leaf discs are aseptically cut into pieces. These pieces are transferred onto tissue regeneration medium supplemented with an antibiotic, kanamycin. From the transformed discs shoots grow directly. The cells which do not undergo transformation will die due to kanamycin. Therefore, antibiotics and herbicides should be used carefully because even in low concentration many cells are damaged. When regeneration has accomplished, selection should be done thereafter. Besides, another difficulty associated with successful selection is the regeneration of shoots from transformed calli because the explants may be heterogeneous and non-transformed cells could not be selected. Therefore, such methods should be used that can ensure escape of only few non-transformed shoots from selection. However, it is ensured by using leaf discs as only the cells which are in direct contact of medium containing antibiotic/herbicide will undergo regeneration.
Table 9.6. Selectable marker genes of vectors and their applications.
||Substrates used for selection
||Gene ble (unknown enzyme)
||Neomycin phosphotransferese (nptll)
||Gentamycin acetyl transferase (gat)
||Hygromycin phosphotransferase (hpt)
||Dihydrofoate reductase (dfr)
||Streptomycin phosphotransferase (spt)
||Mutant form of acetolactase synthase (als)
||Bromoxynil nitrilase (bnl)
||5-enolpyruvate shikimate-3 –phosphate (EPSP)-synthase (aroA)
||PPT (L-phosphinothricin, also called bialaphos)
||Phosphinothricin acetyltransferase (bar)
In addition, there is an alternative procedure where there will be no selection pressure imposed on cells/shoot that develop from explants. In this method, samples of tissue from regenerated shoot are taken, the samples are tested for expression of a marker gene. There is a number of marker genes which are commonly described as reporter genes
or scoreable genes
or screenable genes
(Table 9.7). Some of the reporter genes which are most commonly used in plant transformation are: cat, gus, lux, nptll., etc.
They are briefly discussed as below :(i) Chloramphenicol acetyl transferase (CAT) gene (cat gene)
: The cat
gene is not used as a selectable but as reporter gene. It was first isolated from the bacterium E.coli
but it is absent in higher plants and mammals. In transformed cells its presence can be detected by assaying the enzyme CAT on 32
P-chloramphenicol mixed growth medium. Therefore, the enzyme uses acetyl Co-A-chloramphenicol-P32
as substrate and transfer acetyl CoA to chloramphenicol converting thelater into acetyl chloramphenicol which is detected autoradiographically.
Table 9.7. Examples of some of the reporter genes used as screenable markers.
(ii) Neornycin phosphotransferase (NPTII) gene (nptII gene)
||Substrate / assay
||Detection of acetyl chloramphenical by autoradiography
||Glucuronides (PNPG, X-GLUC, REG, NAG)
||Detection of fluorescence,
||Luc i ferase
||Decan and FMNH2, ATP+O2+luciferin
exposure of X ray film
||Kan+32P-ATP (in situ assay)
||Detection of radioactivity
||Arginine+ketoglutaric acid + NADH
: The npt
IIgene confer resistance against kanamycin and detoxifies it by phosphorylation. It encodes enzyme NPTII. Presence of npt
IIgene in transformed tissue can be detected by selecting them on kanamycin supplemented medium. Similarly, the presence of this enzyme is also detected in transgenic plants or
transformed tissue. Commonly nos
promoter is linked with npt
IIgene so that synthesis of enzyme NPTII may be started well. However, if npt
IIgene has adverse effect on expression of desirable gene, its expression can be improved by using an alternative approach.
Reiss et al.
(1984) have discussed in detail the assay of enzyme NPTII. Firstly, NPTII is fractionated by using non-denaturing polyacrylamide gel electrophoresis (PAGE). In agar layer, radiolabelled 32
P-ATP is used with kanamycin. The gel (that contain the enzyme NPTII) is covered with agar containing both 32
P-ATP and kanamycin. The entire preparation is incubated at 35°C. As a result of phosphorylation of kanamycin 32
P is incorporated into it, the presence of which is detected autoradiographically.
(iii) Luciferase gene (lux gene)
: The lux
gene is found in gloveworm (firefly) and bacteria that secretes the enzyme luciferase. Due to secretion of this enzymes the gloveworm becomes luminescent in dark. The lux
gene has been transferred into tobacco through Ti-plasmid of Agrobacterium.
gene containing bioluminescent tobacco plants were produced.
Similarly a green fluorescent protein (GFP) isolated from the jellyfish, Aequorea victoria
are used as reporter gene or tag in a wide variety of organisms. These act as visible marker for gene expression.
(iv) The b-galactosidase gene (lacZ gene)
: The lacZ
gene that encodes b-galactosidase is a polylinker as it contains several restriction sites but maintains the proper reading frame. Most DNA fragments cloned into polylinker disrupt lacZ
gene and abolish b-galactosidase activity. When a foreign gene fused with lacL
gene is inserted into a microbial cell1
, its presence and function can be detected. When the genetically engineered microbial/plant/animal cells contained a reporter gene is allowed to grow on medium containing a chemical X-gal (i.e.
5-bromo-4-chloro-3-indolyl-b-D-galacto-pyranoside), b-galactosidase hydrolyses X-gal, and releases an insoluble blue dye. The release of dye shows the presence of foreign gene. If there appears no color, it means the gene isdisrupted.