Authors: Sunil Kumar, Om Prakash Patidar and Ashok Yadav
Conventional fruit breeding has had little success in improving fruit crops and is constrained due to long juvenile period, genetic erosion, genetic drag and reproductive obstacles (Varshney et al., 2011). Thus, there is an urgent need for the biotechnology-assisted crop improvement, which ultimately aimed to obtain novel plant traits. Advancement in plant genetic engineering have made it possible to transfer gene into crop plants from unrelated plants and even from nonplant organism. Over the last two decades numerous transformation techniques have been developed for plants. Both Agrobacterium-mediated transformation and microprojectile bombardment based transformation are standard laboratory techniques in plant labs. These biotechnological approaches are a great option to improve fruit genotypes with significant commercial properties such as increased biotic stress resistant or abiotic stress tolerances; nutrition; yield and quality. Currently, public concerns and reduced market acceptance of transgenic crops have promoted the development of alternative marker free system technology as a research priority. Recently, it was demonstrated in apple that transgenic plants without marker genes can be recovered and confirmed its stability by molecular analysis (Malnoy et al., 2010). In 2011, for first time it was described authentically "cisgenic" plants in apple cv. Gala (Vanblaere et al., 2011).
Methods of Plant Transformation
|Direct method||Indirect method|
|Physical gene transfer||Chemical gene transfer||Biological method of gene transfer|
|1. Biolistics||1. PEG-mediated||1. Agrobacterium Mediated|
|2. Microinjection||2.Calciumphosphate coprecipitation|
|3. Liposome mediated||3.DEAE-Dextran procedure|
|4. Silicon carbide fiber- mediated||4.PolycationDMSO technique|
|5. Pollen tube pathway method|
1. Agrobacterium Mediated Transformation:
In this method, A. tumefaciens or A. rhizogenes is employed to introduce foreign genes into plant cells. A. tumefaciensis a soil borne gram-negative bacterium that causes crown-gall, a plant tumor. The tumor-inducing capability of this bacterium is due to the presence of a large Ti (tumor-inducing) plasmid in its virulent strains. Similarly, Ri (root-inducing) megaplasmids are found in virulent strains of A. rhizogenes, the causative agent of “hairy root” disease. Both Ti and Ri-plamids contain a form of “T-DNA” (transferred DNA). The mechanism of gene transfer from A. tumefaciens to plant cells involves several steps, which include bacterial colonization, induction of the bacterial virulence system, generation of the T-DNA transfer complex, T-DNA transfer, and integration of the T-DNA into the plant genome. The process of T-DNA transfer is initiated upon receipt of specific signals (e.g., phenolic compounds) received from host cells.
2. Microprojectile (Particle) Bombardment Transformation:
Microprojectile bombardment is one of the direct gene transfer method for development of transgenics. This method was developed in 1980s to genetically engineer plants that were recalcitrant to transformation with Agrobacterium. Subsequently, the technique has been widely used to produce transgenic plants in a wide range of plant species. The technique involves coating microcarriers (gold or tungsten particles approx. 0.6–1.0 mm in diameter) with the DNA of interest and then accelerating them at high velocities, to penetrate into the cell of essentially any organism. Briefly, the microcarriers are spread evenly on circular plastic film (macrocarrier). The entire unit is then placed below the rupture disk in the main vacuum chamber of the biolistic device. A variety of rupture disks are available that burst at pressures ranging from 450 to 2,200 psi. Below the macrocarrier is a stopping screen, in which a wire-mesh is designed to retain the macrocarrier, while allowing the microcarriers to pass through. The target tissue is placed below the launch assembly unit. Under a partial vacuum, the microprojectile is fired, and helium is then allowed to fill the gas-acceleration tube. The helium pressure builds up behind a rupture disk, which bursts at a specific pressure, thus releasing a shock wave of helium that forces the macrocarriers down onto the stopping screen. The microcarriers leave the circular plastic film and continue flying down the chamber to hit and penetrate the target tissue, thus delivering the DNA.
3. Alternative transformation systems: Transgenics without marker genes:
A highly desirable approach to promote public acceptation for future commercialization of transgenic plants and products is focused on the elimination of marker genes from transformed plants or the direct production of marker-free transgenics (Kraus, 2010). These newly and promising approaches are highly dependent on previously established highly efficient regeneration protocols that may be based on organogenesis or embryogenesis (Petri et al., 2011). There are various technologies such as homologous recombination, cotransformation, site-specific recombination (Cre/loxP site specific recombination system, R/RS system, FLP/FRT system etc.) or marker elimination by transposons to remove selective marker genes (Manimaran et al., 2011). However, there are still few marker-free fruit species transformation protocols. Strawberry leaf explants were transformed with site-specific recombinase for the precise elimination of undesired DNA sequences and a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants (Schaart et al., 2004).
MAT (multi-auto-transformation) (Ebinuma et al., 1997) combined with the Agrobacterium oncogene ipt gene, for positive selection with the recombinase system R/RS for removal of marker genes acting as “molecular scissors” after transformation were used as alternative approach in citrus plants (Ballester et al., 2008). Also, in apricot (López-Noguera et al., 2009) a similar strategy was used. Regeneration of apricot transgenic shoots was significantly improved to non-transformed plants (regenerated in non-selective media).
Moreover, it was significantly higher in comparison with previous published data using resistance to kanamycin mediated by nptII gene. The lack of ipt differential phenotype promoted difficulties to assess the excision of the marker genes that require periodic assays. Complete excision of marker genes ranged from 5 to 12 months, however, only 41% of the regenerated transgenic shoots R-mediated recombination occurs correctly. In Citrus sp., it was also reported that anomalous excision of marker genes promoting failures in the expression of the reporter genes. Apple (Malnoy et al., 2010) and pineapple sweet orange (Ballester et al., 2008) transformation using ‘‘clean’’ binary vector including only the transgene of interest were carried out to create marker-free transformants. In plum (Prunus domestica), transformation was carried out without reporter or selectable marker genes using a high-throughput transformation system (Petri et al., 2011).
4. Cisgenesis and multigene transformation:
Other relevant advance in fruit species transformation was the proposal made by Schouten et al. (2006), the “cisgenesis”. This term means the use of recombinant DNA technology to introduce genes from crossable donors plants, isolated from within the existing genome or sexually compatible relative species for centuries therefore, unlikely to alter the gene pool of the recipient species. Cisgenesis includes all the genetic events of the T-DNA as introns, flanking regions, promoters, and terminators (Vanblaere et al., 2011). This methodology proposes to transfer the own plant DNAs, the P-DNAs. The use of this technology requires the construction of whole plant derived vector from the target species. Within the target species genome, it must be a DNA fragment with two T-DNA border-like sequences oriented as direct repeats ideally about 1-2 kb apart with suitable restriction sites for cloning of a desirable gene. Up to 2011 there is no any report of real “cisgenesis” plantlets, in agreement with Schouten et al. (2006) definition of the topic. In 2011, Vanblaere et al. developed apple cv. Gala cisgenic plants by expressing the apple scab resistance gene HcrVf2 encoding resistance to apple scab. Marker-free system was employed for the development of three cisgenic lines containing one insert of the P-DNA after removing by recombination with using chemical induction. These lines were not observed different from non-transformed cv. Gala plants. Cisgenic plants are essentially the same as the traditionally bred varieties, and they might be easier to commercialise than the “problematic” transgenic plants (Schouten et al., 2006). Critical opinions to these proposals also were clearly exposed, the uncontrolled P-DNA integration into the plant target genome can cause mutations or affect to the expression of other native genes, altering the behaviour of that cisgenic plants in an unpredictable manner (Akhond and Machray, 2009).
The multigene transfer (MGT) methodology consist in introducing more than one gene at once. Commonly, most of the transgenic plants are generated by introducing just one single gene of interest, but now MGT are being developed to obtain more ambitious phenotypes as the complete import of metabolic pathways, whole protein complex and the development of transgenic fruit species with various new traits simultaneously transferred. In this sense, this technology would be highly desirable for commercial fruit species cultivars to obtain new traits related with large fruit size, high coloration of the fruit epidermis, flesh firmness and virus resistance (Petri et al., 2011) at the same time without the need of several rounds of introgressive backcrossing.
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Malnoy, M., Boresjza-Wysocka, E. E., Norelli, J. L., Flaishman, M. A., Gidoni, D., and Aldwinckle, H. S. (2010). Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene. Tree genetics and genomes, 6(3):423-433.
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Vanblaere, T., Szankowski, I., Schaart, J., Schouten, H., Flachowsky, H., Broggini, G. A., and Gessler, C. (2011). The development of a cisgenic apple plant. Journal of biotechnology, 154(4): 304-311.
Varshney, R. K., Kailash, C., Bansal, K. C., Aggarwal, P. K., Datta, S. K. and Craufurd, P. Q. (2011). Agricultural biotechnology for crop improvement in a variable climate: hope or hype? Trends in Plant Science, 16: 363-371
About Author / Additional Info:
Author is a PhD Scholar in Division of Fruits & Horticultural Technology, ICAR- Indian Agricultural research Institute, New Delhi, India