Gene transfer in cereals is becoming established at the backdrop of recombinant DNA technology. Until recently, gene transfer in plants was accomplished only through certain conventional methods like sexual hybridization. The entire scenario of transformation was changed after the advancement of genetic engineering.
Utility of Agrobacterium tumeifaciens as biological vector was found to be successful among dicots. However, its potential utility in gene transfer extended to cereals, which constitute major bulk of crop production, have shown poor response. Thanks to the effective and competent nature of the cells and inducers in dicots for facilitating entire process of gene transfer via co-ordinate role of vir proteins.
Presently, cereal transformation is due mainly by employing physical or direct method of gene transfer. Recently, several promising results were shown that Agrobacterium mediated gene transfer could also be the choice for transformation in certain cereals.
Repeated failure of Agrobacterium mediated process in many cereals has led to the reassessment of direct DNA uptake for transformation process. Protoplast is an ideal choice for gene transfer among cereals.
Some of the key advantages of protoplast among cereals systems are:
(i) DNA concentration can be manipulated in order to ensure that genes can reach and enter every protoplast.
(ii) Complete status of cells may appear due to wound response triggered during enzymatic and mechanical isolation of protoplast. Complete status of cells required for efficient regeneration programme.
(iii) Access for foreign gene to every component cell is possible. Thus, increasing choice of recovery of transgenic plants from entire cell population.
(iv) Protoplast mediated gene transfer can bypass the usage of biological vectors that can overcome host range barriers.
Initial evidence on the stable transformation of cereals stems from direct gene uptake into the protoplast of wheat i.e., Triticum monococcum. The gene construct involves PBR322 derived plasmid, maize transposable elements, nos promoter NPT marker gene.
Plasmid DNA and protoplast were mixed in presence in PEG. After culture for several days dividing cells were embedded and colonies were selected and finally possession of NPT II enzyme activity was noticed. Transformation frequency was recorded only about 1 in every 106 protoplast.
In earlier studies, direct gene transfer to cereals using protoplast was found to be a reasonable success. The stable integration of the transferred DNA and transmission of characters to subsequent generation in Mendelian inheritance pattern was observed.
Protoplast of forage grass Lolium monococcum and Triticum monococcum were transformed by applying electric pulse. Transformation was achieved by interaction of protoplast with plasmid pABD1 carrying NPT II selectable marker gene and Cam 35S promoter.
PEG mediated uptake was used for better uptake and transformation. Heat shock treatment of 45°C for 5 min followed by 10 S at 0°C enhances uptake process. In sugarcane, however, using same plasmid pABD1, efficiency of heat shock treatment did not show any improvement in transformation. Protoplast obtained from rice cells have been reported to be transformed by PEG mediated direct uptake.
Agrobacterium in Cereal Transfer:
It has been emphasized earlier that Agrobacterium utility is restricted to dicotyledons. However, following success of Agrobacterium mediated transfer in rice, several comprehensive investigations and strategy has been extended to wheat. Further studies have shown that only a few wheat cultivars exhibit favourable transformation response to binary vector of Agrobacterium.
Most of the cultivars still show reluctancy for transformation. Interestingly, a super binary vector was created by introducing additional copies of vir gene from Agrobacterium strain, A28131, which is known for its higher degree of transformation. In addition, modification of polyamine ratio (spermidine) in regeneration media, significantly enhance successful transformation in wheat.
Agroinfection in Cereals:
Agroinfection were carried out in maize plants by inoculating virus with strains of Agrobacterium carrying randomly repeated copies of DNA of Gemini maize streak virus their T- DNA. From the studies, ability of Agrobacterium to transfer T-DNA in the maize plants raised apprehension about limited host range of this bacterium.
Further exploration of Agrobacterium carries repeated copies of other Gemini viral DNA in its T-DNA in its T-DNA to other cereals was stabilized. Similarly, wheat Drawf virus, a member of the Gemini virus has been cloned and reintroduced into wheat protoplast. Confirmation on the replication of the viral DNA in wheat as well as maize protoplast opens new avenues in cereals gene transfer.
Gene transfer by pollen mediated process has been studied in few members of cereals, especially in maize. Horst (1987) transformed pollen with chimeric genes containing promoter region and nopaline synthase and NPT II for kanamycin resistance.
Expression of this gene was associated with lowest frequency. Overall transformation of cereals would provide a unique opportunity to study the regulation of foreign gene expression in cereals.
Simple vector construct carrying nos promoter, NPT II and terminator sequence from Cam 35 were introduced into the protoplast and screening was based on kanamycin selection. Transformation frequency was on higher side upto 2% of NPT II activity. Southern blot hybridization indicated the presence of foreign gene in cell colonies.
Electroporation strategy had been explored among cereals with considerable success. Electroporation is a favourable choice and is successfully used for transient and stable formation of cereals. Transformation efficiency in cereals can be manipulated by DNA concentration, the amplitude and duration of electric pulse and also composition of the electroporation medium.
Transformation efficiency was found to be increased drastically in forage grass Lolium and sugarcane protoplast subjected for electroporation using pABD1 plasmid construct. Electroporation mediated gene transfer was 100 times as efficient as that obtained previously with a combined chemical and physical method.
It is however, established that cereal protoplast transformation efficiency depends on refinement of electroporation apparatus like design, circuit and manipulation of initial field strength. Overall, electric field pulse to introduce foreign gene into the plant cells is exemplary.