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One of the significant developments in agriculture biotechnology is the successful transformation of plants by Agrobacterium as a vector. This has formed a basis for the better performance of plants. Existence of close proximity between plant and Agrobacterium leads to the exploitation of these bacteria for the production of transgenic plants.
In addition, accumulation of substantial amount of information regarding molecular mechanism of T- DNA has contributed significantly in gene transfer process. The research work on T-DNA transfer process is dynamic and close to completion.
The ability of Agrobacterium to transfer DNA into plant cells and expression of their DNA in plants pave the real concept that this process could be exploited for the insertion of any desirable piece of DNA into the plant cell by replacing T-DNA. Although none of the genes or DNA sequence within the T-DNA sequence is essential for the transfer process, its 25 bp border sequence is indispensable and retained during disarming of Ti plasmid.
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Dicot vs. Monocot:
Several evidences have shown that Agrobacterium mediated transformation of dicotyledonous plants are more efficient than monocotyledonous plants. Several reasons were quoted on this issue. Competency of plant system in tumour formation is crucial for transformation. The window of competency and wound response among plants establishes ideal atmosphere for transformation.
Both plant and its tissue differ in their wound response. It is established that plants with a prominent wound response develop larger populations of wound adjacent competent cells for regeneration and transformation. Several plants are recalcitrant to Agrobacterium transformation and do not express appropriate wound responses.
Cereals are generally difficult to transform. The complete failure to transform cereal plants with Agrobacterium is probably due to the lack of proper wound response. However, a few among monocot plants with their appropriate wound response are able to transform with Agrobacterium. Among dicots, members of Leguminosae have been recalcitrant to transformation with Agrobacterium and also due to lack of proper wound response.
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Appropriate wound response can be elucidated as induced de-differentiation of wound- adjacent cells and accumulation of competent cells. Wounding of cereal tissue does not trigger de-differentiation of wound-adjacent cells. Instead, they accumulate phenols and dies. This is probably one of the key reasons why it is impossible to transform cereals with Agrobacterium.
However, only one report has been evidenced on the transformation of cereal cell culture with Agrobacterium. Due to insufficient data and lack of convincing evidence, it would be considered as artifacts. Recently several published reports supported cereal transformation.
Design of Agrobacterium Transformation Vectors:
Plant transformation requires construction of non-oncogenic vectors. Hence, disarming of Ti plasmid is indispensable at this stage. Presence of oncogenes in T-DNA region is not essential for target gene transfer.
Therefore, dislodging of oncogenes by replacing gene of interest is an essential criterion in vector construction. There are two types of Agrobacterium vectors suitable to carry essential genes to plant have been designed. Most of the transformations are carried out by using either binary vector or co-integrative vector.
Binary Vector:
Designing of binary vector is based on broad host range plasmids. One of the highlights of binary vector is that it can replicate in dual systems such as E. coli and Agrobacterium. Binary vector can be maintained in Agrobacterium without the process of co-integration into the Ti plasmid. Binary vector is otherwise called as Shuttle vector.
Essentially binary vector contains both border sequences, marker genes and many unique restriction sites for the insertion of target gene between T-DNA borders. In addition, antibiotic resistant genes are present for selection in E. coli and Agrobacterium. Some binary vectors contain only one border sequence.
The other component of binary vector contains helper Ti plasmid or disarmed Ti plasmid that provides vir gene products in transferring gene of interest. Once gene of interest is inserted in binary vector, it is then then mobilized from E. coli to A. tumeifaciens preferably through triparental mating (Fig. 14.10) or through electroporation. The disarmed Ti plasmid in Agrobacterium synthesize vir gene products that are involved in transfering target gene present between two border sequences from bacteria to plant cell.
Co-integrative Vector:
In this approach, the vector is designed to co-integrate into the T-region of the Ti plasmid. In the construction of one of the co-integrative vectors (PGV3850), the entire T-DNA of the nopaline plasmid, except right and left border and also nopaline synthase gene, was replaced by PBR322 sequences, into which another PBR322 containing gene of interest (X gene) can recombine. A single recombination event via PBR322 leads to integration into the Ti plasmid.
In the construction of variant type of co-integrative vector, the entire T-DNA of the nopaline plasmid, for example, Ptic58Tre, except border sequences and nos gene was replaced by PBR322 sequence. The gene of interest (X gene) is introduced in the PBR322 sequences. Co-integrative vector consists of several components like origin of replication of E. coli, plant selectable marker gene such as NPT II, PBR322 homologous sequence and right border.
The co-integrative vector containing X-gene is then mobilized into Agrobacterium. The disarming of Ti plasmid of Agrobacterium can be accomplished by removing T-DNA except left border. Disarmed Ti plasmid retains vir genes as well as PBR322 homologous sequence. Once co- integrative intermediate vector is mobilized into Agrobacterium with disarmed Ti plasmid; both plasmid vectors undergo recombination into the single co-integrative systems.
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Introduction of PBR322 homologous sequence on both the vector share common sequence for homologous recombination, where a single recombination event leads to integration into the Ti plasmid. Following recombination, co-integrative vector becomes single as defective plasmid. Expression of vir genes then cut T-region encloses with X-gene processed and transported into plant cell (Fig. 14.11).
Use of pMNON205:
A different line of approach and the most well designed version of co-integrative vector exist in the form of split end vector (SEV). In this approach, the border sequence, particularly right border and oncogenes are completely removed from Ti plasmid. A small part of the T-DNA referred to as Limited Internal Homology (LIH) and left border is retained.
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This type of approach is used in the split end vector (SEV) systems. Another plasmid vector to be introduced into Agrobacterium also enclosed with LIH region, as well as right border. Following mobilization of this plasmid into Agrobacterium, homologous recombination takes place between the two plasmid vectors. The co-integrated DNA reconstruct contains functional border sequences.
This type of transformation vector is widely employed in the transfer of many genes into the plants. Stability of co-integrative plasmid is one of the key highlights of this vector. Binary vectors on the other hand are not stable in Agrobacterium in the lack of drug selection. Transformation experiments with tomato and other plants evidenced that utility of co-integrative system results in higher frequency of transformation than binary vector.
