ADVERTISEMENTS:
This article provides an overview on genetic engineering of plants.
By the conventional plant breeding techniques, significant achievements have been made in the improvement of several food crops.
These age- old classical methods, involving gene transfer through sexual and vegetative propagation, take very long time.
ADVERTISEMENTS:
For instance, about 6-8 years may be required to develop a new rice or a wheat by sexual propagation. Rapid advances in gene structure and function, coupled’ with the recent developments made in the genetic engineering techniques have dramatically improved the plant breeding methods to yield the desired results in a short period.
Plant genetic transformation technology basically deals with the transfer of desirable gene(s) from one plant species to another (or insertion of totally new genes) with subsequent integration and expression of the foreign gene(s) in the host genome. The term transgene is used to represent the transferred gene, and the genetic transformation in plants is broadly referred to as plant trans-genesis.
The genetically transformed new plants are regarded as transgenic plants. The development of transgenic plants is the outcome of an integrated application of recombinant DNA (rDNA) technology, gene transfer-methods and tissue culture techniques.
Why Transgenic Plants?
It is only through the recombinant approach (genetic engineering) of biotechnology, new genes with desired characters (that may or may not be present in other plants) can be introduced into the plants. Further, it is possible to manipulate the existing genes to make the proteins with suitable alterations e.g. increase in the content of an essential amino acid.
ADVERTISEMENTS:
The most important reasons for developing transgenic plants are listed:
i. To improve agricultural, horticultural or ornamental value of plants.
ii. To develop plant bioreactors for inexpensive manufacture of commercially important products e.g. proteins, medicines, pharmaceutical compounds.
iii. To study the action of genes in plants during development and various biological processes.
Genetic Traits Introduced into Transgenic Plants:
Since plant cells are totipotent (i.e. a single plant cell can regenerate into a whole plant), the genetically engineered cells with new gene(s) can produce a transgenic plant. This plant carrying the desired trait will give raise to successive generations.
Many genetic traits have been introduced into plants through genetic engineering:
i. Resistance to herbicides
ii. Protection against viral infections
iii. Insecticidal activity
ADVERTISEMENTS:
iv. Improved nutritional quality
v. Altered flower pigmentation
vi. Tolerance to environmental stresses
vi. Self-incompatibility
Criteria for Commercial Use of Genetically Transformed Plants:
ADVERTISEMENTS:
For large-scale commercial application of genetically engineered plants, the following requirements have to be satisfied:
i. Introduction of desirable gene(s) to all plant cells.
ii. Expression of cloned genes in the appropriate cells at the right time.
iii. Stable maintenance of new gene(s) inserted.
ADVERTISEMENTS:
iv. Transmission of new genetic information to subsequent generations.
Promoters and Terminators:
The ultimate objective of gene transfer is its correct expression to provide the desired character/ trait. The appropriate expression of genes is made possible by the presence of promoters and terminators. The DNA sequence upstream the coding region is the promoter while the terminator is the sequence at the 3′ terminus.
The promoters are responsible for the commencement of transcription whereas the terminators ensure the ceasation of transcription at the correct position. Promoters possess certain inherent characters such as promoter strength, tissue specificity and developmental regulation which determine the efficiency of promoter function in gene expression.
Agrobacterium-derived promoters and terminators:
ADVERTISEMENTS:
The genes coding for napaline synthase (nos) in Ti plasmid of Agrobacterium are frequently used as promoters and terminators in plant transformation vectors. Originally derived from bacteria, the genes coding for opine synthesis are well adapted to function in plants. In fact, nos promoter is regarded as constitutive by many plant biotechnologists.
The 30S promoter of CaMV:
The cauliflower mosaic virus (CaMV) 30S promoter is the most widely used as a promoter in plants. This promoter, (a 30S RNA gene) is expressed in almost all the tissues of the transgenic plants. The driving strength of 30S promoter is much higher in dicots compared to monocots.
The 30S promoter is preferred for the appropriate expression of selectable marker genes and reporter genes. In recent years, the efficiency of 30S promoter has been further increased by suitable modifications in the enhancer region.
Promoters for monocots:
Since 30S promoter is not very efficient for monocots, alternates are used. Ubiquitin I promoter or the rice actin promoter are frequently used for high expression of transgenes in monocots.
ADVERTISEMENTS:
Inducible promoters:
In recent years, certain inducible promoter systems for transgene expression have been developed. These promoters differ from 30S promoter which is constitutive in nature.
There are mainly three inducible expression systems:
1. Non-plant-derived systems:
These inducible promoters are independent of the normal plant processes. A specific exogenous chemical will induce their expression, e.g. tetracycline, ethanol, steroid, copper. Non-plant-derived inducible promoters are useful, but not economically viable.
2. Plant-derived systems based on response to environmental signals:
ADVERTISEMENTS:
These systems are not independent, since they are plant-derived and actually form a part of normal plant processes. Plant-derived systems respond to a variety of environmental signals e.g. -wound, heat shock.
3. Plant-derived systems based on developmental control of gene expression:
Certain genes that express at a particular stage of plant development have been identified. The promoters of some of these developmental genes are useful for transgene expression, e.g.
i. Senescence-specific gene expression
ii. Abscisic acid inducible gene expression
iii. Auxin inducible gene expression
Although plant-derived systems (2 and 3) are not independent as non-plant derived one (1), they are still useful in plant biotechnology since no exogenous inducer is required.
Tissue-specific promoters:
Efforts are on in recent years to isolate promoters that can drive gene expression in a tissue specific manner. The advantage with tissue-specific promoters is that the expression (which may produce a harmful compound) is confined to selected tissues that are not consumed by humans or animals. In fact, some tissue-specific promoters have been isolated, and are in use.
Transgene Stability, Expression and Gene Silencing:
The genetically transformed plant cells are grown in vitro to finally regenerate to plants. At the initial stages of plant cell growth, some evidence for the success of plant transformation can be obtained e.g. resistance to antibiotics, herbicides etc.
The integration of transgenes with the host plant genome can be confirmed by some molecular techniques—Southern hybridization, polymerase chain reaction. This is usually carried out by analysing the seeds at T1 generation.
What is actually required for the success of plant transformation is the efficient and stable expression of transgene, and not just its presence in plant genome. It is just useless to have a desired gene without appropriate expression.
Scaffold Attachment Regions and Gene Stability:
Scaffold attachment regions (SARs; also known as matrix attachment/associated regions, MARs) are the regions of DNA isolated based on their ability to bind to the nuclear scaffold (protein depleted chromosome forms a central scaffold surrounded by DNA). SARs are known to stabilize gene expression in transgenic plants. During the course of transformation, SARs are ligated to the flanking regions of foreign gene. It is observed that the transformation efficiency by particle bombardment is increased by use of SARs.
The presence of SARs along with transgene stabilizes or normalizes gene expression, e.g. SARs from yeast and tobacco have helped to stabilize gene expression in plants. The degree of stabilization of genes by SARs is dependent on the affinity of SARs towards the nuclear matrix of the target plant cells. SARs are particularly useful for stabilizing the gene expression when the copy number of DNA introduced is high. In the absence of SARs, high DNA copy number would result in an instable and highly variable gene expression.
Introns and Gene Expression:
The presence of introns between the promoter and coding regions of a gene will significantly influence the gene expression. The gene expression is enhanced when introns are used in monocotyledonous plant species. For instance, introduction of introns between cauliflower mosaic virus 35S promoter and β-glucuronidase significantly improved gene expression in maize. It is believed that introns may stabilize mRNAs and increase the protein biosynthesis.
ADVERTISEMENTS:
There are conflicting reports on the effect of introns on the gene expression in dicotyledonous plants. Some workers have reported stimulation while others contradict this claim. It is now accepted that the intron-containing constructs will enhance gene expression in transgenic monocots, although the picture is less clear as regards dicots.
Gene Silencing:
There are in fact many instances of gene transfer that are not properly expressed. The instability of gene expression (or inadequate gene expression) in transgenic plants is referred to as gene silencing. The mechanism of gene silencing is not well understood although it is predominantly due to the phenomenon of homology dependent gene silencing (HDGS). Two types of gene silencing are known in plant biotechnology-—transcriptional gene silencing and post-transcriptional gene silencing. The two differ at the level of gene expression where silencing occurs.
Transcriptional gene silencing (TGS):
When the transgenes share homology in their promoter regions, TGS occurs. It is due to altered methylation patterns and altered chromatin conformation. Gene silencing occurs by repression of transcription.
Post-transcriptional gene silencing (PTGS):
Sometimes, the expression of a homologous transgene may inhibit the expression of the transgene and endogenous gene. This primarily occurs by decreasing the stability of RNA, and thus a reduced expression. This is often observed when strong promoters are used to drive the transgene expression which may result in co-suppression or PTGS, and thus a lower level of gene expression. For this reason, some workers prefer to use weak promoters rather than strong promoters.
Post-transcriptional gene silencing is due to the production of double-stranded RNA either in the nucleus or cytoplasm. The double-stranded RNA is formed when antisense RNA is produced due to the activity of RNA dependent RNA polymerase.
Strategies to avoid gene silencing:
The control of gene silencing is not an easy job, since it occurs in an unpredictable fashion. Some general recommendations, are, however, made to minimize the impact of gene silencing in transgenic plants
i. Reduction in the number of transgenes inserted (i.e. reduced copy number).
ii. Avoiding the use of promoters and transgenes with high degree of homology.
iii. Minimizing/avoiding the use of multiple copies of the same promoter or terminator.
Gene silencing is not observed in chloroplast (plastid) transformation, hence it is preferred in recent years.
