The below mentioned article provides a study note on transgenic plants.
The application of recombinant technique for horizontal transfer of gene in plant system has several limitations. Plant genome is exceedingly complex, involving a huge amount of DNA, which can accommodate sequences equivalent to 6-7 million genes in an average crop plant. Several genes are located outside the nucleus, namely the chloroplastids and mitochondria.
Moreover, every gene, in any system, has its promoter, structural sequences and terminator—all of which require to be identified before introduction. For instance, the nitrogen fixing gene of bacteria—Rhizobium has 17 genes of 24 kilo-bases, distributed under eight operational units or operons.
All these units require be identifying and isolating, along with their promoter and terminator for introduction. The other limitation in the plant system is the absence of a large number of vectors, which would carry the desired gene to the recipient. The most commonly used vector is the Ti plasmid of Agrobacterium.
However, despite these limitations, the progress achieved so far with the existing technology can be considered as moderate (Table 24.2). In almost all major crops, for crop improvement, some of the agronomic traits could be introduced by transgenic approach where the genetic variability has been utilized from other gene pool. Here, some of the traits improved through transgenic approach are being discussed.
I. Resistance to Biotic Stresses:
Resistance to biotic stresses can be categorized under following headings:
1. Insect Resistance
2. Virus Resistance
3. Disease Resistance
1. Insect Resistance:
Transgenes either from microorganism or from plant or other origin can be introduced into plants to increase the level of insect resistance:
a. Bacillus thuringiensis (Bt) encodes for the Bt toxin which are active against Lepidopteran insect has been introduced in many of the crop plants like rice, maize, fibre yielding plant cotton, vegetables like tomato and potato against many insects.
b.Cowpea Trypsin Inhibitor (CpTi) from cowpea, α-amylase Inhibitor from Phaseolus vulgaris, Lectins from Galanthes nivalis (snowdrop)—all these different kinds of proteinase inhibitor genes give resistance against some insect pests. Transgenic tobacco, pea and potato have been obtained carrying these genes show resistance against insects.
2. Virus Resistance:
A number of coat protein genes from different virus groups have been found to provide resistance by inhibiting the virus replication of the challenge virus during the infection.
Many of the transgenic tobacco, potato, tomato, soya-bean, rice, maize have been produced using the coat protein gene of TMV (Tobacco Mosaic Virus), PVX (Potato Virus X), TYLCV (Tomato Yellow Leaf Curl Virus), BMV (Bean Mosaic Virus), RSV (Rice Strip Virus), MDMV (Maize Dwarf Mosaic Virus), etc.
Some antiviral protein or ribosome inactivating protein (RIPs) from different plant sources have been identified to give the resistance against virus. Transgenic tobacco and potato have been produced with PAP (Pokeweed Antiviral Protein) are found to be resistant to TMV.
3. Disease Resistance:
A large number of plant defense response genes encoding antimicrobial proteins are now have been cloned.
The products of these defense response genes may include
(i) Chitinase and PR proteins,
(ii) Ribosome inactivating proteins (RIPs),
(iii) Antifungal proteins (AFPs),
(iv) Enzyme for phytoalexin,
(v) Phenolics, Lectins, etc. Here is a list of transgenic plants generated in various crops for resistance against fungal and viral diseases:
II. Resistance to Abiotic Stresses:
Several abiotic stresses such as drought, salinity and extreme temperatures have a common consequence of causing cellular water deficit or osmotic stress. Some osmoprotectants within cells like proline, soluble carbohydrates like polyols and some ammonium compounds like glycine, betaine help to lower the osmotic potential and maintaining turgor within the cell.
Here is a list of few transgenic plants derived against resistance to abiotic stresses:
III. Resistance to Herbicide:
Transgenic plants against various herbicides such as phosphinothricin (bialaphos), glyphosate, sulfonylurea, imidazolinones, bromoxynil, atrazine, 2, 4-D, etc. have been generated in different food crops, vegetables, horticultural and ornamentals species. Herbicide resistant transgenic plants like cotton, flax, canola, corn and soya-bean have already released.
The following genes are used from different origin, active against different herbicides.
IV. Transgenics for Quality:
1. Transgenic for Improved Storage:
Transgenic tomato ‘Flavr Savr’ with delayed ripening developed by Calgene, UBA using antisense RNA technology of ACC deaminase. Monsanto produced transgenic tomato to express a gene from Pseudomonas which produces metabolites other than ethylene from ACC. Another gene SAM hydrolase from bacteriophage T3 also reduced the ethylene production in tomato giving more shelf life.
2. Transgenic Flowers have Longer Shelf Life:
Transgenic carnations expressing antisense ACC oxidase and producing flowers with little ethylene and marked delay in senescence.
3. Transgenics for Flower Color and Shape:
Delphinidin is the compound responsible for blue colour in flower, the Flavonoid 3′-5′ hydroxylase (F 3′-5’H) is the enzyme responsible for blue colour formation has been cloned and transgenic violet carnation “Moon-dust” is available. Modification of intensity of flower color from white to pink has been obtained by antisense RNA technology of chalcone synthase and transgenics have been obtained in Petunia carnation, Gerbera, etc.
4. Transgenics for Nutritional Quality:
Tailor made starch i.e. starch with reduced level of amylose and higher amount of starch have been developed in potato. Trehalose is an additive which improves the taste quality in processed food. Transgenic tobacco has been produced by Calgene producing trehalose. Over-expression SPS (Sucrose Phosphate Synthase) holds promise for achieving the sugar content increase, transgenic tomato has been produced with over-expression of SPS.
Considerable progress has been made in the improvement of nutritional quality of legume storage protein expression. Brazil nut 2S albumin protein gene has got expressed in Arabidopsis, rapeseed, soya-bean, French bean and potato. The soya-bean glycine gene and also the lysine have been introduced into rice to increase the protein content and also to make it more digestable.
To save a large population of children from vitamin A deficiency, the improved variety of transgenic rice have been developed capable of synthesizing the early inter- ‘ mediate Geranyl-Geranyl diphosphate (GGPP) which can be used to produce the uncolored carotene phytoene by expressing the enzyme phytoene synthase in endosperm.
The transgenic ‘Golden Rice’ has been obtained by introducing the whole P-carotene synthesis pathway related genes into rice endosperm capable of producing P-carotene, the precursor of vitamin A synthesis. The iron content increase is possible with the use of ferritin transgene from Aspergillus fumigatus, transgenic rice plants have been produced with two fold increase in iron content and high activity of phytase.
V. Transgenics for Agricultural Traits:
1. Transgenics for male sterility and production of hybrid seeds (using sterility- fertility system): The character of male sterility has wide use in securing fertile hybrids in interspecific hybridisation. This character has now been widely used for transgenesis to secure male sterile transgenic plant. This method involves induction of male sterility-fertility restoration.
Bacterial coding sequence for ribonuclease (barnase) has been attached with anther specific promoter which will kill the pollen grain by production of cytotoxic element in tapetal cells. Another gene which is a ribonuclease inhibitor (barstar) gene has also been isolated from the same bacteria and used to produce transgenic plants that nullify the effect of barnase gene.
The transgenic plant with barstar gene was developed which was crossed with the male sterile transgenic plant with (barnase) gene. The F1 progeny is restored due to suppression of cytotoxic ribonuclease activity in the anther. The transgenic male sterile line already has been obtained in Brassica.
2. Transgenics for improvement of uptake capacity of different nutrients from soil by introducing the transporter gene which reduce the dependence on fertiliser application.
3. Transgenics for good harvest index is attempted using the phytochrome genes by regulating the flowering or fruit development at a time.
4. Transgenics for seedless fruit i.e., without pollination the ovary may mature into fruit with high pulp quantity.
5. Transgenics for terminator seed production technology: A lethal gene (RIP)— Ribosome Inhibiting Protein interferes the synthesis of other proteins in the plant cell, which prevents the germination of seeds when the gene is attached to a particular promoter and expressed only in seed.
As a result the desired traits will be expressed in first generation seeds only, not in the successive generations. This mechanism of terminator seed production will help always to maintain the pure line by companies which has marketed it.
VI. Transgenic Plants as Bioreactors:
1. Transgenic Plants for PHB Synthesis:
Transgenic plants are also being produced to be used as bioreactors for synthesis of various types of chemicals; the technique is called as Molecular pharming. Polyhydroxy Butyrate (PHB), an polyester with thermoplastic property, is synthesized by transgenic Arabidopsis after introducing the transgene from Alcaligenes euthophus.
2. Transgenics for Production of Vaccines and other Peptides:
The use of transgenic plants as the producer of many peptides of pharmaceutical use and the vaccines are becoming significant now-a-days. Human Serum Albumin (HSA) is being successfully produced in transgenic tobacco and potato plants.
Transgenic maize plants have been produced capable of producing avidin. Potato tubers have also been used as the bioreactor for high level production of a recombinant single chain FV (SCFV) antibody.
The edible plant vaccine against diarrhoea expressed in potato, but as it is cooked before eating, heat may denature the vaccine protein. The vaccine protein gene is now have been introduced into banana which can be eaten raw. Transgenic tobacco plant is capable of producing HBV (Hepatitis B virus) surface protein.