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Genetic Transformation of Rice: Problems, Progress and Prospects | OMICS International
ISSN: 2375-4338
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Genetic Transformation of Rice: Problems, Progress and Prospects

Saroj Kumar Sah*, Amandeep Kaur, Gurwinder Kaur and Gurvinder Singh Cheema
School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana 141 004, Punjab, India
Corresponding Author : Saroj Kumar Sah
School of Agricultural Biotechnology
Punjab Agricultural University
Ludhiana 141 004, Punjab, India
Tel: +91- 9779569724
Received November 06, 2014; Accepted December 11, 2014; Published December 14, 2014
Citation: Sah SK, Kaur A, Kaur G, Cheema GS (2014) Genetic Transformation of Rice: Problems, Progress and Prospects. J Rice Res 3:132. doi: 10.4172/2375-4338.1000132
Copyright: ©2014 Sah SK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Plant genetic engineering has become one of the most important molecular tools in the modern molecular breeding of crops. Now a days, production of transgenic plants is a routine process in some crop species. Transgenes are delivered into plants to confer novel traits such as improving nutritional qualities, resistance to pests. It is possible to insert genes from plants at evolutionary distant from the host plant, as well as from fungi, viruses, bacteria and even animals. Genetic transformation requires penetration of the transgene through the plant cell wall, facilitated by biological or physical methods. Over the last few decades, a significant progress has been made in the development of new and efficient transformation methods. Despite a variety of available DNA delivery methods, Agrobacterium and Biolistic mediated transformation remain two predominantly applied approaches. The objective of this article is to review the currently used methods for genetic plant transformation, their biological requirements and critical parameters

Genetic transformation rice; Agrobacterim; Biolistic transformation.
Cereals are the most important source of calories to humans since rice, wheat and maize provide 23%, 17% and 10% calories globally [1]. Rice (Oryza sativa L.) is a well-known economical cereal, because it is a staple food included in the diet and most important source of food for more than half of the population. It has been reported that more than 90 per cent of the world’s rice is grown and consumed in Asia, where it is a major source of income to rural people [2,3]. Adoption of green revolution varieties led to a drastic change in rice production. Between 1996 and 2011, the population of the low-income countries grew by 110 per cent, but rice production increased by 180 percent only from 257 million tons in 1996 to 718 million tons in 2011. Despite these advances in rice production, still 800 million people is not getting food every day. It is estimated to increase up to 25 per cent of rice production by the year 2030 [1]. This additional rice must be produced from good lands without opening up more fragile lands for rice cultivation. Agriculture production is decreasing due to biotic and abiotic stresses. The major abiotic stresses worldwide which cause threat to food security are high salinity, drought, submergence and cold [4-8]. Among these abiotic stresses, salinity is one of the major factors restricting productivity of crop plant worldwide [9]. Area under salt stress is increasing due to certain factors like climate change, rise in sea levels, excess irrigation without proper drainage in islands and underlying rocks rich in harmful salts,. If current scenario will continue, 50 per cent of present cultivated land will be lost for agriculture by 2050 [10]. Therefore, abiotic stresses are one of the main concerns to fulfill the required food demand [11]. Rendering the problem will entail development of rice varieties, which have higher yields, excellent grain quality, and resistance to biotic and abiotic stresses.
Many breeding programs have been conducted to increase the environmental stress tolerance, pest and disease resistance to the crop. Although conventional breeding programs such as Conventional hybridization, hybrid breeding, wide hybridization and ideo type breeding have developed some rice varieties in addition to several lines have been released in the Philippines, Bangladesh and India [12] but the success rate of conventional breeding is not sufficient [12,13] to meet the requirements. So there is a need to develop rice varieties that can withstand high levels of salt, drought and water stress with optimal yield levels
Therefore, it is of utmost important to augment the productivity of rice to cope with the increased threat of population boom which is expected to reach 9.1 billion by 2050. To combat these issues, various methods of conventional breeding programs have been deployed, albeit with little progress. In this aspect, genetic transformation can be integral tool in breeding strategies. It permits access to an unlimited gene pool through the transfer of desirable genes [14]. The development of plant transformation techniques during the past decades has made it possible to improve crop plants by introduction of cloned genes. The two most critical steps to be the master for transformation of plants are to transfer of foreign DNA into the plant cell and regeneration of plants from transformed cells [15]. The callus induction and regeneration in tissue culture of rice depend upon different factors like genotype, type of explants and media supplement like basal salts, organic component and growth regulator [16-19]. Development of genetically engineered plants with enhanced tolerance to abiotic and biotic stresses is the important challenges in plant biotechnology research. Rice transformation is a major goal in cereal biotechnology, because rice is world’s most important food crop and it is also known as model of cereal genomics [20].
In early 1980s, genetic transformation of crop plants i.e. based on recombinant DNA technology was started and this offered an advantage of transferring novel genes across taxonomic boundaries unlike conventional breeding [3]. Genetic engineering has been used as a prominent tool for rice improvement. Although gene transformation in japonica rice is performed routinely in several laboratories, but the system in indica rice is more complicated [15]. Until now, the number of copies of a gene(s) inserted and chromosomal locations of the integrated genes are not controllable, the expression of the introduced genes varies among individual transformants. Therefore, a relatively large number of transgenic plants must be developed in order to select desirable transformants as well as to study the expression of introduced genes [21].
The most commonly used method for transformation are Biolistic approach and Agrobacterium mediated transformation. This review will summarise various gene delivery methods applied to improve rice traits. Subsequent molecular analysis of the transgenic rice will also be discussed. Additionally, it will consider the future prospects of transgenic researches on the crop.
Genetic Transformation
Purpose of genetic transformation
Main purpose of genetic transformation is to generate plants with useful phenotypes i.e. unachievable by conventional plant breeding, to correct faults in cultivars more efficiently than conventional breeding and to allow the commercial value of improved plant lines to be captured by those investing in the research more fully than is possible under intellectual property laws governing conventionally bred plants. Some reasons for genetic modifications are yield improvement, more resistant to disease and pest resistance, herbicides tolerance, better nutritional value, increased shelf life, better climatic survival by increasing tolerance to drought, flood or frosty conditions to allow the use of previously inhospitable land, higher crop yields, reduced farm costs, increased farm profit and improvement in health and environment..
Biological requirements for transformation
The essential requirements in a gene transfer system for production of transgenic plants are availability of a target tissue including cells competent for plant regeneration, a method to introduce DNA into those re-generable cells and a procedure to select and to re-generate transformed plants at a satisfactory frequency.
Methods of Genetic Transformation
Agrobacterium mediated genetic transformation
The soil pathogen Agrobacterim tumefaciens has been extensively studied since 1907, when it was identified as the causative agent of crown gall disease [22-24] Braun initially proposed the Agrobacterium as a source of a ‘tumor inducing principle’, possibly DNA, that permanently transformed plant cells from a state of quiescence to active cell division.
A. tumefaciens is a soil dwelling bacteria that naturally infect dicots and causes tumorous growth resulting in crown gall disease. Tumor formation results from incorporation of T-DNA (transfer DNA), a part of small independent DNA molecule outside the bacterial genome called Ti (tumor inducing) plasmid. Phenolic compounds exuded from plant wounds that stimulate the expression of vir genes, located on Ti plasmid and responsible for its excision, transfer and integration into plant genome. The natural capability ofAgrobacterium was manipulated in plant transformation by replacing the genes causing tumorous growth by genes of interest [25]. However,Agrobacterium has a natural tendency to infect dicot plants, whereas, monocots including wheat were considered recalcitrant to Agrobacterium transformation. Therefore, most of the Agrobacterium-mediated transformation procedures were established for dicots, whereas, monocots including important cereals such as wheat, rice and maize lagged behind for a considerable time [26-28]. Low frequency of T-DNA transfer into the target genome was the major limitation. Nonetheless improvements of co-cultivation conditions, use of acetosyringone, selection and regeneration methods for transformed tissues and incorporation of super binary vectors have helped in extending the host range of Agrobacterium to several monocots including important cereals.
Agrobacterium approach is the most popular method to deliver genes to plant cells because of its clean insertion, low-copy number of the inserted genes, easy to handle, higher efficiency, more predictable pattern of foreign DNA integration. Studies on A. tumefaciens provided the basis that has made this soil bacterium as dominant customer for plant transformation.
Critical parameters to increase the efficiency of Agrobacteriummediated genetic transformation are: Concentration of Agrobacterium broth (in terms of OD), time of inoculation, time of co-cultivation, concentration of acetosyringone.
Optical density of Agrobacterium culture must be in the range of 0.4 to 1.00. During the Agrobacterium infection, OD600 of the bacterial suspension must be adjusted within 1.0 because high concentrations of bacteria caused serious injury to callus thus lowering the transformation efficiency.
Co-cultivation duration (5-25min), exposure the explants to 0.4 OD Agrobacterium for 10 min was found to be optimum.
Co-cultivation period should be between 2 to 5 days. Co-cultivation for 2 days showed better infection without browning or Agrobacterium overgrowth.
Co-cultivation with filter paper increased transformation frequency because the filter paper prevented overgrowth of bacteria. In co-cultivation medium, use of cysteine reduces browning of the calli, and Acetosyringone as well as glucose to induce the vir gene activity. Concentration of acetosyringone should be between 100-150 μM. But higher concentration of acetosyringone (200 μM) in co-cultivation media resulted in lower frequency of regeneration likely due to necrotic effect of acetosyringone.
Biolistic Process (Particle Bombardment) mediated genetic transformation
Biolistic transformation was initially invented in 1987 by American Geneticist, Associate Professor John C, Sanford from Cornell University where he and his colleagues showed that small micro projectiles could be delivered into a cell without killing it. In this approach (Also called gene gun or particle bombardment), microscopic particles coated with the DNA fragment representing the desired gene are shot into the plant cells using a special device called gene gun. The key principle behind the gun is to accelerate particles by explosion. A small portion of the DNA which enters the cells becomes incorporated into the chromosomes of the plant cell. The gene gun technique helps to overcome some of the deficiencies of Agrobacterium method such as bacterial contamination, low-efficiency of transfer to cereal crops, and inconsistency of results. The tungsten was first used or DNA coating later on it was replaced by gold particles because tungsten could be oxidized easily and it was potentially harmful to the cell.
It has many advantages than other techniques like rapid gene transfer, efficient, non-specific to tissue, complex cloning strategies with no biological constraints or host limitations and simultaneous multiple gene transfer. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene co-transformation is straightforward. It has bigger advantage that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This approach can be used for transfer of more than one gene simultaneously in a host plant. As many as 14 genes have been co-introduced in rice by this approach [29]. Nowadays, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation [30]. A genotype independent method for rice transformation was originally reported by Christou et al. [31] and it has been widely used throughout the world. Researchers at the International Rice Research Institute, Philippines, have used particle bombardment successfully to transform over 20 different cultivars adapted to different eco-geographic conditions. These cultivars have been transformed with a range of agronomically important genes like psy, crt1, cry, ferritin, FRO2, Xa21, Bt, Chitinase,enod12, PEPC, glgC, rolC, sd1 confirming the genotype independence of the transformation method (For details see Table 1) [32]. The main disadvantages of particle bombardment in comparison with Agrobacterium i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods.
Genetic transformation occurs in the two stages: DNA transfer into the cell followed by DNA integration into the genome. The integration stage is much less efficient than the DNA transfer stage, with result of that only a small proportion of the cells that initially receive DNA actually become stably transformed. In many cells, DNA enters but expressed for a short time (transient expression), but it is never integrated and it is eventually degraded by nucleases. Transient expression occurs almost immediately after gene transfer, it does not require the regeneration of whole plants, and it occurs at a much higher frequency than stable integration. Therefore, transient expression can be used as a rapid assay to evaluate the efficiency of direct DNA transfer and to verify the function of expression constructs. In addition, transient expression following particle bombardment with a reporter gene such as gusA or gfp is used routinely to compare different expression constructs and to identify those with most appropriate activities. When the aim is to extract recombinant proteins from transgenic plants, transient expression following particle bombardment may also be used to produce small amounts of protein rapidly for testing [63]. Hoffman et al. [64] used particle bombardment for the mechanical transmission of poleroviruses and particle bombardment is routinely employed for the inoculation of whole plants and leaf tissues with viruses that are difficult to introduce via conventional mechanical infection. It also has an important role in extending virus-induced gene silencing (VIGS) into economically important crop plants [65].
Critical parameters to increase transformation efficiency
Type/physiological and developmental stage of the target tissue: Embryogenic calli of rice have been extensively used in particle bombardment experiments. The calli should be young not more than 30 days old. If we use old calli, the transformation efficiency will be low as calli loses its regeneration capacity ultimately affecting transformation efficiency.
Particle type/size, helium pressure and target distance on bombardment: Efficiency of the DNA delivery to target cells depends largely upon the size of micro-particles and the force in which these DNA coated particles are pushed towards the target cells. The depth of penetration into the target tissues can be controlled by varying particle size, helium pressure and the target distance. The target distance has direct relation with target area i.e. lesser the target distance narrow is the target area and greater the target distance, wider is the target area. So these three parameters are important in determining optimal conditions for efficient DNA delivery to target cells/tissues.
Effect of particle and DNA loads: For obtaining high efficiency of DNA delivery to target tissues, different DNA to particle ratios has been used to determine a suitable combination. A particle load of 3 mg (0.5 mg/shot) is sufficient to produce high level of transient GUS expression during conjunction by 9 μg of DNA (1:3 particle-DNA ratio).
In-Planta transformation
A number of advances in reducing the dependence to tissue culture have been made in rice transformation including, in planta transformation method. Cereal transformation via the tissue culture phase has been successful, but involves several limitations. The use of tissue culture allows selection of single transformed cells which are regenerated in a whole plant. However, the tissue culture approach causes somaclonal variation due to both epigenetic effects and chromosomal rearrangements [66,67]. The in planta transformation method overcomes the disadvantages of the conventional in vitro Agrobacterium-mediated transformation method. The latter requires sterile condition, that is time consuming and causes somatic mutation or somaclonal variation in plant cells during in vitro culture, and some plants are recalcitrant to regeneration. In contrast, in planta transformation involves no in vitro culture of plants cells or tissue, which is its greatest advantage.
Floral dip transformation
Clough and Bent [68] modified the Agrobacterium vacuum infiltration method to transform Arabidopsis thaliana. This process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 μlL-1 of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were produced transformed progeny at the highest rate. Plant tissue culture media, the hormone BAP and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately two fold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates two fold.
Desfeux et al. [69] also investigated the mechanisms that underlie the floral-dip method for Agrobacterium-mediated transformation in Arabidopsis to facilitate its usage in other plant species.In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Their results suggested that ovules were the site of productive transformation in the floral-dip method, and further suggested that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure.
Rod-in et al. [70] reported Agrobacterium-mediated transformation method for rice carrying the gusA gene to infect rice spikelets via the floral-dip method. The tip-cut spikelets of the rice inflorescence stage 51 (beginning of panicle emergence: tip of inflorescence emerged from sheath) were dipped in the Agrobacterium and co-cultivated at 25 °C for 3 d. Floral dip approach transform primarily anthers and to a small extent the ovary. The highest transformation efficiency in case of anther was 89.16% whereas in case of ovary 7.23% Their results suggest that floral-dip transformation is a simple potential tool for production of transgenic rice with no requirement of tissue culture.
Naseri et al. [71] demonstrated the inoculation of A. tumefaciens into embryonic apical meristem of the soaked seeds, a region on the seed surface where a shoot would later emerge and was pierced twice up to depth of about 1 to 1.5 mm with a needle (0.70 mm) dipped in the A. tumefaciens inoculums. The inoculated seeds were then placed on filter papers on wet per liter in flasks covered with aluminum foil and incubated at 23°C in dark for nine days that 70 to 75% of inoculated seeds are germinated to seedlings. PCR results showed that 24% plants inoculated with Agrobacterium integrated transgene
Critical parameters: During the agrobacterium infection, OD600 of the bacterial suspension must be 1.0 for higher transformation.
Source of Explants
Tissue culture which involves manipulating the totipotent nature of plant cells is one of the necessary technology for production of transgenic plants. For genetic transformation in different experiments, many types of explants has been used by researchers likewise mature seeds, mature zygotic embryos, immature embryos, mature embryos, calli, inflorescence, embryogenic apical meristem, spikelet, roots, isolated shoot apices (Tables 1-3).
Vectors and Markers in Gene Transfer
In plant research, binary vectors are standard tools for delivery of a wide range of genes into the cells of higher plants [72] This vector has certain important characters like multiple cloning sites, bacterial origin of replication, unique restriction sites and selectable markers gene cassettes enabling the recognition of untransformed cells. A selectable marker gene encodes a product that allows the transformed cell to survive and grow under conditions that kill or restrict the growth of non-transformed cells. The most used genes in rice are dominant selectable markers that confer resistant to antibiotics or herbicides i.e. ble (glycopeptides binding protein), dhfr (dihydrofolate reductase), hpt (hygromycin phosphotransferase), nptII (neomycin phosphotransferase), bar and pat (phosphinothricin acetyltransferase), csr1-1 (acetolactate synthase), tms2 (indolaacetic acid hydrolase) (For details see Table 1 [20]. In addition to selectable marker genes, plant researchers also used reporter genes known as screenable marker, a visual marker or a scorable marker,such as gus A (ß-glucuronidase, GUS), luc (firefly luciferase), gfp (green fluorescent protein) (For details see Table 1) [20]. It generates a product that can be detected using a simple and often quantitative assay. It is mainly used for confirming transformation, determining transformation efficiency and monitoring gene or protein activity. It has ability to form fusion genes at the transcriptional level and it can be used to assay the activity of regulatory elements. It also forms translational fusion products, which allows them to be used, to monitor protein localization in the cell or at a whole-plant level. ß-glucuronidase activity can be easily monitored within 1-2 days post co-cultivation in infected plant tissues by enzymatic conversion of colourless X-Gluc substrate to blue precipitates [73] so, it is a powerful assay for detecting transformation events.
Next major aspects of binary vector is the usage of constitutive promoters providing transgene expression in majority of the plant tissues. Studies have been done on rice transformation by using CaMV35s constitutive promoters. By using this promoter Brietler et al. [74] reported 70-80% genetic transformation for yfp and hph genes. Besides this promoter, there are many others has been used for genetic transformation in rice such as Actin 1, ubi-1, ubiquitin, OSCc1 (Tables 1-3).
Transgenic Analysis
To confirm the real transgenic, efforts should be undertaken with a long term goal rather than just the analysis of the initial transformants. Development of transgenic plants is not only limited to the confinement of laboratory, it should have wider scope of application. So, to test the real transgenic we have to go for different complementary tests like GUS Assay, PCR, Semi-quantitative PCR, Quantitative Real Time PCR, Southern blotting, Western blotting, ELISA, Northern blotting. Most of the studies conducted molecular analysis of the primary transformants, rather than proceeding further. GUS analysis revealed transformation efficiency in wide range (7.23% to 95%) that has been shown by different studies by using different explants in various cultivars of rice [21,75- 77]. PCR analysis showed various range of transformation efficiency i.e. 16%-100% [48-53]. Molecular analysis were carried out by most of the workers to check the integration pattern and copy number by southern blot analysis, northern and real time pcr [78,79,81-84].
Future Prospects
As we look to the future of rice biotechnology, we have many transformation technique but till now no transgenic line of rice has been released successfully. Near about 100 genotypes have been used by different groups for transformation (Tables 1-3). Among them, Taipei 309 (Japonica rice) and Basmati (Indica rice) has been widely used in Agrobacterium-mediated genetic transformation with an efficiency of 31% and 40-75%,respectively.Though some reports has been shown that some of the transgenic line has been released. Gupta et al. [85] reported that Huaghi-1 Rice for insect resistance has been released by Huazhong Agricultural University, China. SO, we have to focus on particular assay of that transgene which has been transferred along with series of confirmatory test. Ultimately we need high yield so, our main objective should be to check our transgene is working or not, which may not affect the yield. As for example, if we transfer salinity tolerance gene, we must go for salinity screening technique, in which transgenic line must show higher tolerance level in respect to wild. In most of the research this assay are lacking. So, to check or confirm real transgenic we must go for particular assay.
In the present scenario, population growth is increasing day by day along with demand for food supply. Genetic transformation must be implemented to bridge the gap between production and human need. Whatever achievement has been done so far in transgenic, is not sufficient to fill the demand. The full realization of the plant biotechnology revolution depends on successful and innovative research, as well as on favorable regulatory guidelines and public acceptance. Thus, all the strategies discussed in the present review will definitely contribute to biotechnological breeding programs of rice for its improvement.



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