From the early past to the present, biotechnologies have produced the ability to genetically transform a wide variety of plant species. The plant transformation technologies have changed the face of agriculture and plant biology. Plant genetic transformation is one of the key technologies for crop improvement in addition to emerging approach for producing recombinant proteins in plants. Both plastid genomes and plant nuclear can be genetically modified. Until now, essential functional differences between the prokaryotic-like genome of the plastid and the eukaryotic genome of the plant cell nucleus will have an impact on characteristics of transgenic organism. Thus, the main goals are to generate transgenic plants with the traits of interest as well as minimizing the amount of transgenic DNA in plants while maximizing stability of gene expression and trait performance. In this review, two broad groups of gene delivery methods will be discussed namely, (bilogical and physical methods) and subsequently there applications for improving disease resistance will be discussed.
Fundamental element for crop improvement and development of new cultivars with desirable characters is the ability to modify the genetic composition of a plant. To develop new lines by sexual hybridization, plant breeders have employed the naturally happening genetic variability in existing germplasm [
To generate genetic variability, the development of somatic cell hybridization and protoplast culture is one of the first patterns. Additionally, in vitro culture of plant cells in suboptimal conditions is established to induce genetic variations which term somaclonal variation [
The first step for genetic transformation is the DNA delivery to cells or tissues, followed by culture and selection to permit only those tissues and cells having a marker gene (for instance, herbicide resistance gene or antibiotic) to proliferate and survive further. Then, plants are regenerated from the surviving cells, embedded and rooted in the soil. Such primary transformants after that are used for molecular analyses for determination of copy number of the transgenes of interest [
Approaches for delivering DNA into plant cells and gene transformation can be divided into two major categories: direct and indirect DNA deliveries. Direct method does not employ bacterial cells as mediators, as an alternative, it uses a chemical alteration or physical force such as electric discharge or pressure to deliver the vector DNA into a host cell [
Agrobacterium tumefaciens is a Gram-negative soil phytopathogenic bacterium which causes crown gall disease in plants and it can be grown in vitro in simple culture media without any added plant growth hormones and the bacterium [
The understanding of the T-DNA integration pathways have considerably enriched by releasing new studies and in the same time, it exposed many host factors that participate in these events [
Briefly, the following steps of host genetic transformation facilitated by Agrobacterium are: The induction of Agrobacterium’s virulence machinery results in expression and activation of the virulence genes (vir genes), some of these genes are important in the transfer of T-DNA from the bacterium to host cell, whereas others helps in targeting T-DNA to the nucleus and most likely to the precise integration site in the host cell for T-DNA [
This method has been found to be challenging due to the low copy number and large size of Ti plasmids, leading to difficulties in plasmid manipulation and isolation, and it limited in the range of plant species that can be transformed because not all tissues or species are susceptible to Agrobacterium [
Beside some drawbacks for Agrobacterium transformation, this method still works in many labs and high transformation rate frequencies could be obtained. More recently, by group of researchers Chetty, Ceballos [
Particle bombardment is a theoretically simple powerful tool for biotechnologists allowing direct gene transfer to a broad range of cells and tissues which have been found difficult to transform by other technique, and is more valuable for improving species which have high level of heterozygosity such as potato and cassava [
The term biolistic was established to describe the nature of the delivery of the foreign DNA into living cells,and the development of the particle bombardments from concept to useful process was first invented by Sanford [
Principally, the particle bombardment device comprises of a mechanism to accelerate the particles to desired speeds and adjust their penetration into the receiver cells. Initially, the gun powder discharge device was used to accelerate inert metal microprojectiles coated with biologically active compounds [
Agrobacterium Strain | Transgene copy number (%) | |||||
---|---|---|---|---|---|---|
nptII | uidA | |||||
1 | 2 | >2 | 1 | 2 | >2 | |
AGL1 | 52 | 24 | 24 | 52 | 29 | 19 |
EHA105 | 67 | 21 | 12 | 62 | 25 | 13 |
GV3101 | 52 | 33 | 15 | 55 | 31 | 14 |
MP90 | 76 | 24 | 0 | 67 | 33 | 0 |
via metal screen, the optimized helium gas pressure forces the microprojectiles to deliver biologically active compounds and penetrate the target tissue to affect gene transfer [
The critical factors to maximize transformation efficiency by this system are the delivery of optimal amount of DNA with minimal injury to the receiver plant tissue, size and density of microprojectiles, attachment of DNA to microprojectiles, helium pressure at which microprojectiles are propelled and distance between rupture disc are all important factors [
One of the preferred methods for both transient gene expression studies and stable genetic transformation for improvement of crops is biolistic gene transfer [
More recently for biolistic transformation of sugarcane Xiong, Jung [
respectively and they assumed that all three DNA coating protocols are similarly effective for stable genetic transformation of sugarcane (For more details, see
Recently, progress of a new active transgenesis method based on particle bombardment would be more effective than common particle bombardment and integration of multiple copies of DNA could be decreased [
Wu, Du [
Delivery of biomolecules mediated by mesoporous silica nanoparticleis (MSNs) particularly interesting because without the assistance of protein transport systems, proteins are often unable to cross the barrier of cells membrane [
In general, the low activity of endocytosis and the barrier of cell walls limit the application of nanotechnology on plant systems. Up to now, only calcium phosphate nanoparticles and poly dendrimer (DNA vector),showed that particles could be entered into plants through simple co-culture methods and works as carriers for gene delivery without any additional assistance, and high transient delivery efficiency could be obtained [
Treatment | Experiment | Number of bombardments | Number of transgenic plants | Number of transgenic plants/bombardment* |
---|---|---|---|---|
Spermidine | I | 8 | 13 | 1.63 |
II | 8 | 15 | 1.88 | |
III | 14 | 25 | 1.79 | |
Spermidine total | 30 | 53 | 1.76 ± 0.07 | |
Protamine | I | 8 | 23 | 2.88 |
II | 8 | 9 | 1.13 | |
III | 14 | 16 | 1.14 | |
Protamine total | 30 | 45 | 1.71 ± 0.58 | |
Seashell DNA del TM | I | 8 | 7 | 0.88 |
II | 8 | 9 | 1.13 | |
III | 14 | 25 | 1.79 | |
Seashell DNA del TM | 30 | 41 | 1.26 ± 0.27 |
More recently, to develop plant transient gene expression system, Mou, Chang [
The use of silicon-mediated transformation first reported by Kaeppler, Somers [
Although the attributes of this method have been recognized for being simple, less resource requiring for DNA delivery into plants cells and cost effective [
Moreover, by group of researchers in Pakistan [
The application of electroporation has a major impact on genetic manipulation of organism. In this method, the foreign DNA migrates through high voltage resulting in formation of pores in the plasma membrane [
Practically, high electric fields are applied to protoplasts or cells suspended in a liquid culture medium enclosed in a discharge chamber [
However, there was an attention in bypassing the protoplasts for electroporation because the regeneration of fertile transgenic plants system has not been developed in most plant species [
The first stable transformation through this method was with rice seeds cultured (2) days prior which has been subjected by electro-injection with a plasmid having neomycin phosphotransferase II (NPTII) gene and then transgenic rice were regenerated through somatic embryogenesis [
Recently, resistance against Leaf Spot under improved form of CaMV 35S promoter by over expressing rice chitinase-3 gene in transgenic peanut through Agrobacterium mediated gene transfer was found [
More than 60% of regenerated plantlets were observed whereas transgenic healthy plants with over 42% transformation frequency were formed and all plants were normally grown. Subsequently, by infection with the microspores, (T1) plants were tested for resistance against Leaf Spot. Transgenic strains showed a higher resistance than the non-transgenic plants. Their results indicated that the lines with high enzyme activity similarly showed a high resistance against leaf spot (see
Tissue culture and transformation were the most difficult task for developing the transgenic papaya [
However, genetic engineering through particle bombardment and Agrobacterium-mediate both create relatively random integration of the transgene into the host genome [
Transgenic line | RT-PCR result | Oschit activity | Enhanced chitinase activity | Resistance to C. arachidicola | Correlation to hypothesis |
---|---|---|---|---|---|
Golden untransformed | _ | 1 | _ | _ | _ |
09-G1 | + | 6.5 | Y | Y | √ |
09-G2 | + | 3.5 | Y | Y | √ |
09-G7 | + | 6.2 | Y | Y | √ |
09-G7.3 | + | 3.2 | Y | N | × |
09-G9 | + | 1.9 | N | N | √ |
09-B4 | + | 2.1 | N | Y | × |
09-B5.4 | + | 6 | Y | Y | √ |
09-B13 | + | 2.3 | Y | N | × |
10-B2 | + | 1.8 | N | N | √ |
10-B3.4 | + | 3 | Y | N | × |
Researchers were thinking about more attractive technique to control disease resistance for papaya. Therefore, genetic resistance within the species may be more explained with the advantage of fully first sequenced genome of transgenic papaya [
Development of gene transfer systems in plants probably one of the most challenging aspects of plant research. Now day, the two methods of choice are definitely the Agrobacterium-mediated and the biolistic-mediated DNA delivery systems. From two decades ago, development of transformation technology was viewed primarily as an objective to the production of transgenic crops with improved agronomic characteristics for enhanced crop productivity [
Therefore, gene transfer methods to plants will continue to receive renewed interest in the future. The development of nanoparticles for DNA delivery into plant cells is emerging [
It is a great pleasure that we can take this opportunity to send thanks and appreciation to Dr. Nawroz Abdul Razzak who were available every times and supported us by providing valuable advices. We would also like to point out the contributing role that the University of Sulaimani has played in establishing and growing the knowledge of all lecturers and supported us all of the times. Finally, we would like to express our gratitude for the sacrifices of Peshmarga and their bravery in the fight against IS.
Ahmad HamaAmeen Rashid,DjshwarDhahir Lateef, (2016) Novel Techniques for Gene Delivery into Plants and Its Applications for Disease Resistance in Crops. American Journal of Plant Sciences,07,181-193. doi: 10.4236/ajps.2016.71019