Plants represent the principal source of human foods and livestock feeds and efforts to improve them in many agronomic aspects have focused on plant breeding. The biotechnology revolution in the past decade made possible for plant breeders access new sources of genetic variability for the development of superior cultivars. It has been possible to define additional strategies for crop improvement through the introduction and stable integration of heterologous genes in plant cells with the knowledge of the regulation of the important agronomic characteristics. The genetic manipulation of plants allows their adaptation to different environmental stresses, whether biotic or abiotic. Currently, the production of genetically modified plants occupies a prominent place in both, basic and applied plant research. Genetically modified crops are generated through a process known as genetic engineering, in which genes of interest are transferred to plants without the need of natural crossing. The most widely used methods for introducing transgenes into the genome of plants are
Maize is one of the most cultivated cereals in the world. The main maize producer’s countries are the United States, China, and Brazil, followed by Mexico, France, Argentina and India. Among the big losses faced by agriculture are the attacks of pests and diseases. For maize, these problems have worsened since 1990 because of the increase of the cultivated areas in both the normal growing season and the off season, mainly due to intensive cultivation of maize in the irrigated areas, and lack of adoption of crop rotation in certain fields. In recent years, diseases that were not a problem, increased in importance such as the viruses. Among the strains of the virus complexes, potyviruses cause significant losses in grain and forage of maize susceptible genotypes. Plants have different mechanisms for protection against invasion by pathogens, and different genes directly related to tolerance to viruses have been described in maize. Works have been published using methods of obtaining plants resistant to viruses by antisense, co-suppression and, more recently, RNA interference (RNAi).
This chapter reviews methodologies that have been used to introduce the RNAi construct in maize cells, such as
2. RNA interference
RNA interference is a natural phenomenon of which double stranded RNA (dsRNA) activates a mechanism that degrades complementary RNA in the cell. This process has been described in many organisms such protozoa, flies, nematodes, insects, mouse and human cells (Napoli et al. 1990; Hammond et al., 2001; Agrawal et al., 2003; Baulcombe, 2004; Tang and Galili, 2004) and has been referred as cellular defense against viruses and post-transcriptional regulation of gene expression. In maize, there are extensive reviews done by McGinnis (2009) describing the application of this process as a reverse genetic tool.
Before the identification of the RNAi phenomenon, there were other methods such as T-DNA insertion, transposon elements and physical and chemical mutagens and antisense suppression to generate gene loss-of-function. These approaches, which have been used until today have allowed scientist study the function of many gene or gene families. The earliest version of gene silence was the process called antisense, which involves the introduction of the antisense strand of RNA to silence an internal RNA homologue (Knee and Murphy, 1997). The antisense strand once inside the cell binds to the target RNA by complementation preventing it to be translated. One possible explanation, on that time, was the inability of the ribosomes bind to the dsRNA. Another possible explanation that came up later, was that the dsRNA might also be a substrate for the DICER/RISC an enzymatic complex responsible for degradation of dsRNA in the RNAi process. The first description of the RNAi phenomenon was done by Fire et al. (1998). This group introduced sense and antisense RNA strands in
3. Application of RNAi to obtain transgenic maize lines tolerant to the SCMV
Fuchs and Grüntzig (1996) observed that Sugarcane Mosaic Virus (SCMV) and Maize Dwarf Mosaic Virus (MDMV) were the most important potyviruses, causing significant losses in grain and forage of susceptible maize genotypes. In Germany, the maize fields with mosaic symptoms were first found in the early 80’s (Fuchs and Kozelska, 1984). Since then, MDMV and SCMV have been regularly observed in maize producing regions of Germany, where epidemiological studies have shown the prevalence of SCMV(Fuchs et al., 1996). For the tropical conditions observed in Brazil, it were described three viruses in maize: (i) mosaic which can be caused by four distinct potyviruses transmitted mechanically and, by
The particles of the potyvirus causing the mosaic disease are flexible and have a length of approximately 750 nm and width varying from 13 nm (MDMV and SCMV) to 12 nm for the Johnsongrass mosaic virus (JGMV) (Shukla et al., 1994). Like most plant viruses, the potyviruses have a genome consisting of sense strand RNA-positive, with a length of approximately 10,000 nucleotides and a protein (Vpg) connected to the terminal 5 'genome (Figure 2).
Plants have different mechanisms for protection against invasion by pathogens such as physical barriers, secondary metabolites and antimicrobial proteins. Once established, elicited molecules produced and released by the pathogen induce new defenses such as cell wall strengthening, phytoalexin production, synthesis of proteins related to plant defense, among others. The identification and application of these mechanisms is one of the most effective manners to rapidly improve crop resistance to diseases. Microarray experiments have shown hundreds of genes regulated by plant-pathogen interactions, most of these are defense-related proteins (PRs) or system acquires resistance (SARs) (van Loon et al., 2006).
An alternative strategy for obtaining materials resistant to pathogens, specifically virus, was published by Grumete et al. (1987), when they over expressed part of the genome of the pathogen in a plant and showed a significant increase in resistance. The explanation given at the time was that the disfunction of the gene products derived from the pathogen could inhibit the pathogen. Similar work has also demonstrated the expression of the coat protein of TMV (Tobacco Mosaic Virus) in the generation of resistant tobacco plants (Abel et al., 1986). These plants in the presence of the virus showed no symptoms or showed a delayed onset of symptoms. Additional experiments showed immediately that the level of transgene expression was correlated with the level of expression of resistance (Fitchen and Beachy, 1993, Powell et al., 1990).
Many different types of viruses in plants have been shown to encode silencing suppressors. Suppressors of silencing of these viruses interfere with different steps of processing the RNA silencing present in plants and are important defense responses (Ratcliff et al., 1999). This process was one of the most evident in plants to identify viruses that have proteins that interfere with the system of the plant RNA silencing. In 1998, Anandalakshmi and collaborators and Brigneti and collaborators shown that HC-Pro protein of TEV and 2b of CMV could have this role. A classic paper demonstrated that the inhibition system of the 5' end corresponding to proteins P1 and Hc-Pro was efficient to obtain plants resistant to Plum pox virus (PPV) in tobacco (Di Nicola-Negri et al., 2005). In this same study were tested four regions of the virus genome: (i) nucleotide (nt) 1-733 of the protein corresponding to P1; (ii) nt 954-1603 corresponding to the end of the protein P1 and protein portion of Hc-Pro; (iii) nt 1680-2386 corresponding to the central part of Hc-Pro/P3 and, (iv) nt 1935 to 2613 corresponding to the end of Hc-Pro protein and part of P3. To access the efficiency of each construct in relation to the resistance of transgenic plants to PPV a large number of transgenics was analyzed by ELISA and, it was shown that 90% of transgenic plants were resistant to PPV. Despite all indicates that the target for this group of RNAi gene constructs are based on the 5 'end (mainly the Hc-Pro) there are works based on positive replication region (Guo and Garcia, 1997; Wittner et al., 1998) or in the 3 'end of the coat protein (Ravelonandro et al. 1992; Palkovics et al. 1995; Jacquet et al., 1998).
4. Maize transformation
The insertion of sRNAi in the plant can be accomplished by different ways such as eletroporation,
Significant progress has been achieved in developing technology for genetic transformation of maize in the last decade. Genetic transformation of maize became nowadays a routine procedure for various genotypes in most public and private laboratories working with this culture. For introducing a siRNA construct in maize is necessary (i) an
In vitroregeneration of transgenic maize cells
The establishment of maize regeneration systems from somatic cells constitutes a prerequisite of utmost importance within the process of transgenic maize plants production. Regeneration of maize plants in tissue culture can occur via organogenesis (Zhong et al. 1992) or somatic embryogenesis, and the last one is the most used method.
Grasses were considered recalcitrant species with regard to establishing of totipotent cultures
Armstrong & Green (1985) introduced the terms of Type I and II callus which are currently used for the classification of embryogenic cultures of maize. Type I callus is composed of hard, compact, yellow or white tissue and usually capable of regenerating plants (Vasil & Vasil, 1981). Type II is soft, friable and highly embryogenic (Armstrong & Green, 1985). Type II callus culture is fast-growing and can be kept for a long period of time without losing their totipotency (Vasil, 1987).
Although Type II calli are the most efficient in the production of transgenic maize, Type I calli can also be used. The occurrence of friable embryogenic Type II callus is not so common, only a limited number of maize genotypes are able to express this phenotype in tissue culture, notably the line A188 (Armstrong & Green, 1985) and the hybrid HiII (Armstrong et al. 1991). With the advancement of the
It is known that, in maize, the initiation of regenerable callus as well as the frequency of regeneration of plants are affected by a genetic component and depend on the genotype used (Hodges et al. 1986; Prioli & Silva, 1989). Through a diallele involving eight cultivars of maize, Beckert & Qing (1984) found significant heritability for initiation of somatic embryogenesis and plant regeneration. The high heritability indicates that both the initiation of callus and plant regeneration can be improved by crossing genotypes recalcitrant to highly responsive genotypes (Hodges et al., 1986). The formation of somatic embryos and regenerative ability are under control of genes located in the genome of maize cells (Hodges et al. 1986; Vinh, 1989). However, the physiological and developmental stage at the time of explant excision, the time of the year and, the specific interactions between genotypes and growing conditions of the donor plant, may modify the expression of genes that control the induction of somatic embryogenesis and plant regeneration (Prioli & Silva, 1989).
4.2. Methods of genetic transformation of maize
The different methods of genetic transformation of maize can be divided into two major groups: direct and indirect methods. Indirect method of genetic transformation uses a bacterium,
4.2.1. Transformation of maize cells using microparticle bombardment
Since most of the monocots are not natural hosts for
The particle bombardment of plant cells with DNA of interest is a direct method of transformation designed to introduce nucleic acids into the genome or plastome of cells (Taylor and Fauquet, 2002). It is a methodology commonly used by laboratories working with plant genetic transformation. It was developed in the late 80's to manipulate the genome of plants recalcitrant to
Several physical parameters correlated with the biolistic equipment such as pressure, macrocarrier and microcarrier flight distance, and vacuum, must be optimized for successful transformation. Besides these parameters, the plant material and the gene of interest which will be used should also be tested in preliminary experiments (Sandford et al., 1993).
From the 90's the microparticle bombardment was used to transform a wide variety of plants, including maize. Gordon-Kamm et al. (1990) and Fromm et al. (1990) were the first groups to report the production of transgenic maize from the bombardment of embryogenic callus. Then, several reports of transformation of maize showed that the particle bombardment is a successful technique for inserting foreign genes into the genome of maize with high reproducibility of results (Brettschneider et al. 1997; Frame et al. 2000).
The main advantages of microparticle bombardment is related to the use of simple vectors and easy handling, plus the possibility of inserting more than one gene of interest into cells efficiently (Wu et al. 2002). Although considered a very efficient method of transforming maize, a possible disadvantage is the occurrence of multiple copies of the gene of interest and complex integration patterns, susceptible to silencing, of gene expression in future generations (Wang and Frame, 2004).
Agrobacterium tumefaciensmediated maize transformation
For several years the transformation of monocots by
To enable the use of
Because it is very large, the Ti plasmid is difficult to manipulate, so binary vectors, which are smaller, able to grow both in
The first maize transformation protocol mediated by
5. Gene constructs for RNAi target genes
Transgenes or genes that are inserted via molecular biology techniques in plants such as maize, are basically composed of (i) regulatory sequences that control gene expression, (ii) the selection marker gene and, (iii) the gene of interest.
The main sequences controlling gene expression are promoters, enhancers, introns and terminators. Promoters are DNA sequences, normally present in the 5 'end of a coding region, used by RNA polymerase and transcription factors to initiate the process of gene transcription (Buchanan et al., 2000). Depending on the ability to control gene expression, the promoters are classified as weak or strong, according to the binding affinity of transcription factors with the promoter sequence (Browning & Busby, 2004). Strong or weak promoters can be further classified as constitutive, tissue and / or organ-specific and inducible. A constitutive promoter directs expression of a gene in all tissues of a plant during the various stages of development. The viral
Enhancers are regions of DNA that bind transcription factors responsible for an increase in transcription of a gene, and consequently by an increase in protein expression. Enhancers can be located before or after the coding region. In the genome, sequences of plant enhancers can be located physically distant from the gene which they are controlling, however because of the packaging of DNA in the nucleus, these sequences are geometrically positioned near the promoter. This position allows for an interaction between transcription factors and RNA polymerase II (Arnosti & Kulkarni, 2005).
Introns are non-coding sequences within a gene that are removed during transcription. Although the mechanisms underlying the phenomenon are not completely clear, the incorporation of introns in genes can increase or decrease promoter activity and the levels of transcription (Chaubet-Gigot et al., 2001). Typically, the intron is inserted between the 3 'end of the promoter and the initial codon of the protein of interest (Liu, 2009). Introns such as the rice actin
The regions 3 'UTRs also known as terminator regions are used to confer greater stability to the mRNA, and to signal the end of the transcript preventing the occurrence of the production of chimeric RNA molecules and consequently the formation of new proteins, if the polymerase complex continues transcribing beyond the end of the gene (Lessard et al. 2002). 3' UTRs sequences used in most gene constructs for transformation of maize include the nopaline synthase gene from
The selection gene is a sequence encoding a protein that when expressed in transgenic cells confer an adaptive advantage. The selection gene is used to identify and select cells that have the heterologous DNA integrated into their genome. Selection genes are fundamental to the development of technologies for plant transformation because the process of transferring a transgene to a recipient cell and its integration into the genome is very inefficient in most experiments, and the chances of recovery transgenic lines without selection are generally very low (Liu, 2009).
Currently, the most used selection markers for the production of transgenic maize are those that confer tolerance to herbicides. Among these, the
In majority the gene of interest is a coding sequence or ORF (Open Reading Frame) of a certain protein that when expressed define a characteristic or phenotype of interest. In other cases, is a gene sequence used to silence gene expression, such as the RNAi technology.
An important aspect regarding the use of RNAi for plant biologists is the ability to decide the target region of the gene that should be used to efficiently produce the dsRNA. In 2002 the company Dharmacon (http://www.dharmacom.com) was the first to develop an algorithm as a tool for rational design of a potent silencing, based on data by Reynolds et al., (2004). Today, there are several companies that have developed algorithms for analysis of gene sequence based on a number of parameters that predispose to more effective use of this technology. Many of these softwares are freely accessible on the Internet:
The new synthesized siRNA can target other RNAs on the basis of sequence similarity. Any RNA that possesses sequence similarity with the original trigger dsRNA may be silenced. This fact may limit the use of RNA silencing in plants due to gene family with high sequence similarity (Miki et al., 2005).
One alternative way to express dsRNA in maize is described as followed. The interested cDNA fragment is initially amplified with primers forward containing the
The transgenic T1 plants arise in the frequency around 1% relative to the original number of explants. The first confirmation of the transgenic is done by spraying leaves with 3 mg/L Finale herbicide (ammonium glyfosinate - AgrEvo Environmental Health, Montvale, NJ). The
The second confirmation of the transgenic is done by PCR using primers specific to the gene construct. To produce high-quality, stable transgenic lines it is necessary to define individuals with a single copy insertion and in homozygosity. This decision is based on the premise that expression of one copy is more stable and reliable than multicopy in the following generations. DNA purified from a single leaves (~100 mg of tissue) of T1 transformed plants is screening in a Southern blot analysis to identify events that possess single copy insertion. DNA is digested with restriction enzyme and subjected to gel electrophoresis. After the transfer of the DNA to the nylon membrane it is hybridized either with the
Recent works at Embrapa Maize and Sorghum (Brazil) obtained SCMV resistant transgenic maize plants by transforming friable callus of maize HiII using a construction based on the RNAi technology (data not published). Previous study on the SCMV gene family identified the region of the coat protein as a conserved region that might be used to produce the cassette to silence the expression of the SCMV virus in maize. Once this fragment from the SCMV genome was choose and isolated, it was cloned twice, in inverted position, into the vector pKANNIBAL containing a spacer, transferred to a binary vector pCAMBIA 3301 containing the ubiquitin promoter and NOS terminator and used to transform maize by particle bombardment as explained above.
The phenotypic evaluation of the transgenic plants was done by inoculation of the SCMV virus complex every week for three consecutive weeks starting in a maize V5 stage. The inoculation was confirmed by PCR and microscopy. From the 20 events obtained 30% of the plants did not show any viruses symptoms and in approximately 46% the symptoms reduces along the plant life cycle. These results indicated that the technique of RNAi based on the Coat protein sequence was capable of generating transgenic maize resistant to the SCMV virus (Figure 6).
Other groups also got similar results, in maize, by induced RNAi-mediated transgenic virus resistance. Bai et al. (2008) transformed maize with an hpRNA expression vector p3301 containing the inverted-repeat sequence of the SCMV Nib gene, and obtained transgenic resistant lines. Also, Zhang et al. (2001) constructed an hpRNA expression vector containing reverted-repeat sense and antisense arms to target the MDMV gene encoding the P1 protein (protease) and used this cassette to transform maize embryonic calli and obtain plants tolerant to MDMV viruses.
In the 60’s and 70’s the world experienced a vast increase in the agricultural productivity based on conventional breeding techniques, intensive use of industrial inputs (fertilizers and pesticides), mechanization and cost reduction of management. In the 21st century, molecular biology techniques have been coupled with the conventional breeding techniques to boost up crops productivity. In the mid 90’s the discovery of the RNAi added a new perspective to the gene regulation. This technology became a powerful tool to understand gene function and to the breeders improve crop varieties such as the development of barley varieties resistant to BYDV (Barley Yellow Dwarf Virus) (Wang et al., 2000), reduce the level of glutenin in rice which is important for patients that are incapable to digest it (Kusaba et al., 2003) and among others, to obtain varieties of banana resistant to BBrMV (Banana Bract Mosaic Virus), a virus that has devastated the Southeast of Asia and Indian.
Some applications of RNAi in plants have relied in non
The SCMV research was granted by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig), Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico (CNPq) and Embrapa Milho e Sorgo. This publication has been funded by “Fundo MP2 Embrapa / Monsanto.