Transgenic Plants
Table 9.5. Transgenic plants produced by different methods.
Plants groups | Transgenic plant species | |
1. | Gymnosperms | Picea glauca |
2. | Angiosperms |
|
(i) Monocots | Asparagus sp., Dactylis glomerata, Secale, cereale, Oryza sativa, Triticum aestivum, Zea mays, Avena sativa, Festulaca arundinacea. | |
(ii) Dicots | 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 | Marker genes | |
1. | Antibiotics | |
Bleomycin | Gene ble (unknown enzyme) | |
G418,Kanamycin, Neomycin | Neomycin phosphotransferese (nptll) | |
Gentamycin | Gentamycin acetyl transferase (gat) | |
Hygromycin B | Hygromycin phosphotransferase (hpt) | |
Methotrexate trimethoprim | Dihydrofoate reductase (dfr) | |
Streptomycin | Streptomycin phosphotransferase (spt) | |
2. | Herbicides | |
Chlorosulfuron imidazolinones | Mutant form of acetolactase synthase (als) | |
Bromoxynil | Bromoxynil nitrilase (bnl) | |
Glyphosate | 5-enolpyruvate shikimate-3 –phosphate (EPSP)-synthase (aroA) | |
PPT (L-phosphinothricin, also called bialaphos) | Phosphinothricin acetyltransferase (bar) |
(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 32P-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.
Reporter genes | Enzymes expressed | Substrate / assay | Identification |
cat | Chloramphenicol acetyl transferase | 14C-chloramphenicol +Acetyl CoA (TLC) separation) | Detection of acetyl chloramphenical by autoradiography |
gus | b-glucuronidase | Glucuronides (PNPG, X-GLUC, REG, NAG) | Detection of fluorescence, colorimetric, photometric |
lacZ | b-galactosidase | b-galactosidase (X-gal) | Colony color |
lux | Luc i ferase | Decan and FMNH2, ATP+O2+luciferin | Bioluminescence on exposure of X ray film |
nptll | Neomycin phosphotransferase | Kan+32P-ATP (in situ assay) | Detection of radioactivity |
nos | Nopaline synthase | Arginine+ketoglutaric acid + NADH | Electrophoresis |
ocs | Octopine synthase | Arginine+pyruvate+NADH | Electrophoresis |
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 32P-ATP is used with kanamycin. The gel (that contain the enzyme NPTII) is covered with agar containing both 32P-ATP and kanamycin. The entire preparation is incubated at 35°C. As a result of phosphorylation of kanamycin 32P is incorporated into it, the presence of which is detected autoradiographically.
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.