Genetic transformation is a powerful tool for plant breeding and genetical, physiological or biochemical research, consequently it is an extremely dynamic field. Transgenic plants are commonly used to complete or substitute mutants in basic research, helping the studies of complex biological situations such as pathogenesis process, genome organization, light reception and signal transduction. In this review, recent approaches for foreign gene introduction (e.g. Agrobiolistics, whole tissue electroporation, in planta Agrobacterium transformation), screening (reporter gene possibilities and performance) and transformant selection (ipt selective marker) are discussed. Transgene expression and mechanisms underlying (trans)gene inactivation are presented. Practical applications of genetically modified plants, field tests and commercial transgenic crops worldwide and in Brazil are listed, as well as the main traits and species modified. Potential uses of transgenic plants for animal compound production, biological remediation and synthetic polymer assembly are also shown.
plant transformation; Agrobiolistics; electroporation; reporter genes; transgenic crops
A transformação genética é um valioso recurso para o melhoramento e para a pesquisa em Genética, Fisiologia e Bioquímica. Plantas transgênicas são usadas para complementar ou substituir mutantes na pesquisa fundamental e para auxiliar em estudos de fenômenos biológicos complexos como patogenicidade, organização do genoma, captação de luz e transdução de sinais. Nesta revisão, são discutidas abordagens recentes visando a introdução, screening e seleção de transformantes, estudos sobre expressão do transgene e uso de plantas geneticamente modificadas. Ensaios de campo e culturas transgênicas comerciais são listadas assim como as principais espécies e características modificadas. O potencial das plantas transgênicas para a produção de compostos animais, remediação biológica e síntese de polímeros é igualmente apresentado.
transformação de plantas; Agrobiobalística; eletroporação; genes repórteres; culturas trangênicas
PLANT TRANSFORMATION: ADVANCES AND PERSPECTIVES
Adriana Cristina Alves; Vera Maria Quecini; Maria Lucia Carneiro Vieira*
Depto. de Genética-ESALQ/USP, C.P. 83, CEP: 13400-970 - Piracicaba, Brasil
ABSTRACT: Genetic transformation is a powerful tool for plant breeding and genetical, physiological or biochemical research, consequently it is an extremely dynamic field. Transgenic plants are commonly used to complete or substitute mutants in basic research, helping the studies of complex biological situations such as pathogenesis process, genome organization, light reception and signal transduction. In this review, recent approaches for foreign gene introduction (e.g. Agrobiolistics, whole tissue electroporation, in planta Agrobacterium transformation), screening (reporter gene possibilities and performance) and transformant selection (ipt selective marker) are discussed. Transgene expression and mechanisms underlying (trans)gene inactivation are presented. Practical applications of genetically modified plants, field tests and commercial transgenic crops worldwide and in Brazil are listed, as well as the main traits and species modified. Potential uses of transgenic plants for animal compound production, biological remediation and synthetic polymer assembly are also shown.
Key words: plant transformation, Agrobiolistics, electroporation, reporter genes, transgenic crops
TRANSFORMAÇÃO DE PLANTAS: AVANÇOS E PERSPECTIVAS
RESUMO: A transformação genética é um valioso recurso para o melhoramento e para a pesquisa em Genética, Fisiologia e Bioquímica. Plantas transgênicas são usadas para complementar ou substituir mutantes na pesquisa fundamental e para auxiliar em estudos de fenômenos biológicos complexos como patogenicidade, organização do genoma, captação de luz e transdução de sinais. Nesta revisão, são discutidas abordagens recentes visando a introdução, screening e seleção de transformantes, estudos sobre expressão do transgene e uso de plantas geneticamente modificadas. Ensaios de campo e culturas transgênicas comerciais são listadas assim como as principais espécies e características modificadas. O potencial das plantas transgênicas para a produção de compostos animais, remediação biológica e síntese de polímeros é igualmente apresentado.
Palavras-chave: transformação de plantas, Agrobiobalística, eletroporação, genes repórteres, culturas trangênicas
A commonly accepted definition of plant transformation is: "the introduction of exogenous genes into plant cells, tissues or organs employing direct or indirect means developed by molecular and cellular biology" (Jenes et al., 1993). However, only integrative events, confirmed by molecular and genetical analyses, are correctly designated as genetic transformation (Potrykus, 1990; Potrykus, 1991; Birch, 1997).
Numerous methods for exogenous gene introduction into plant genomes have been described (Potrykus, 1990; Potrykus, 1991), and they can be classified into two groups: indirect gene transfer - where exogenous DNA is introduced by a biological vector and direct gene transfer - where physical and chemical processes are responsible for DNA introduction. Some of the proposed methods show low efficiency and repeatability, being scarcely utilized. Thus, basically three methods are responsible for almost all existing transgenic plants, namely indirect transformation mediated by Agrobacterium (i) or direct systems using electroporation (ii) and biolistics (iii). Detailed explanations, applications and characteristics of these methods can be found in: Horsch et al. (1984), De Block et al. (1984), Tefter (1990), Zupan & Zambryski (1995) and Sheng & Citovsky (1996) concerning the Agrobacterium system; Fromm et al. (1987), Shillito & Potrykus (1987), Gad et al. (1990), Negrutiu et al. (1990) and Neuhaus & Spangenberg (1990) for electroporation procedures and, Klein et al. (1987), Sanford (1988), Morikawa et al. (1989), Birch & Franks (1991), Christou (1992), Sanford et al. (1993) and Christou (1995) for microparticle bombardment.
Regardless of the transformation method, a previous plant regeneration protocol for the species under investigation and a cloned gene of interest must exist. Foreign genes can be obtained from other plant species or even from different kingdoms, like fungi, bacteria and animals.
Every stable transformation process demands the simultaneous occurrence of two independent biological events, which are: stable insertion of the transgene into the plant genome and regeneration of those cells where it occurred, producing a non-chimeric transgenic plant. The need of both events occurring in the same cell makes it a constraint for higher transformation efficiency. Selection markers and reporter genes are frequently used to detect transformation sites.
Although perfectly established, principal transformation methods often undergo changes or improvements to achieve recalcitrant species transformation or higher frequencies.
Agrobacterium-mediated plant transformation without the need of an in vitro culture stage was achieved by employing in planta inoculation (Bechtold et al., 1993; Chang et al., 1994). In this methodology, young flower buds are inoculated with the bacteria under ex-vitro conditions. When seeds are produced, they are submitted to selection for marker-induced resistance. Despite the ease of this method, only Arabidopsis thaliana stable transformants have been obtained and, differences in the efficiency among ecotypes were observed (Bechtold et al., 1993).
Since the system greatly depends on pathogenesis, basic aspects of Agrobacterium-plant interaction have been studied, such as T-DNA transfer and insertion (Bundock & Hooykaas, 1996), susceptibility of plant genotypes (Nam et al., 1997) and influence of host cell cycle stage on T-DNA transference (Villemont et al., 1997). In addition, technical improvements in order to increase transformation rates, such as employment of particle bombardment to provoke microwounding at the inoculation site - Agrobiolistics (Bidney et al., 1992; Brasileiro et al., 1996; Hansen & Chilton, 1996) and intracellular microinjection of bacteria into regions with regenerative abilities (Escudero et al., 1996) have been developed.
Electroporation is a technique that uses electrical discharges to create reversible pores in the plasma membrane, thus allowing the introduction of foreign DNA. Since the main drawback for electroporation employment is the ability to obtain fully developed plants from protoplasts, whole tissues have been used as explants, like zygotic intact embryos in cowpea (Akella & Lurquin, 1993), common bean (Dillen et al., 1995) and rice (Xu & Li, 1994; Rao, 1995).
Immediate identification is a highly desirable feature of transformed cells, but since most introduced genes are unable to induce phenotypes distinct from the wild type, the utilization of a promptly identified reporter gene in cis or in trans provides a screening tool.
The most utilized reporter gene so far is uidA, which codes for a monomeric stable enzyme- b-glucuronidase (GUS), which catalyzes the conversion of methylumbeliferone glucuronide into methylumbeliferone, forming an easily identifiable blue precipitate. There are many advantages of GUS: it is undemanding to visualize, the enzyme is not easily denatured and the precipitate is stable (Jefferson, 1987). However a disadvantage of the GUS system is that it is a destructive assay, that is, the explant cannot be recovered.
Some plants show variable levels of endogenous b-glucuronidase expression (Kosugi, 1990), which impairs interpretation of GUS assay results. Use of an engineered virus as a gene amplification vector of GUS was proposed by Shen & Hohn (1994). These authors reported up to a four fold increase in blue points, indicating that viral DNA was responsible for reporter gene amplification.
Alternatively, Chalfie et al. (1994), Niedz et al. (1995) and Sheen et al. (1995) suggested "green fluorescent protein (GFP)" as useful reporter gene for plant and animal systems. GFP is a protein produced by a jellyfish, Aequorea victoria, that is able to emit green fluorescence under blue light or UV illumination without any additional substrate. The principal advantages of this reporter gene compared to the formerly used, namely GUS, is the easiness of its visualization. It needs no extra substrate for detection and, most important, is a non-destructive assay (Vain et al., 1998). Very recently, soluble, highly fluorescent variants of GFP were produced (Davis & Vierstra, 1998).
Since transformation is a rare event, selective markers impair the development of a great number of non-transformed cells in favor of transgenic ones. However, the use of a selective agent, which is frequently an antibiotic or a herbicide, retards cell differentiation and shoot development in the explant submitted to a transformation process (Colby & Meredith, 1990). In addition, if another transformation of the same material is needed, the selective marker is no longer effective. Due to these limitations, Ebinuma et al. (1997) proposed a selective marker that unlike the former ones imposes no negative effect on non-transformed cells, instead inducing a different phenotype in transgenic shoots. These authors have proposed a construction based on a chimeric ipt gene, which codes isopentenyl AMP, a cytokinin precursor (phenotype ESP - "extreme shooty phenotype") cloned inside the maize transposable element Ac. Transposon Ac was used to remove the gene ipt from the transformed cells in explants with an ESP phenotype after transformation, since during the transposition process the excised element very rarely reintegrates. If it does, it occurs in a sister chromatid and is lost due to somatic segregation.
Concerning the size of cloning vectors, most of the plasmids employed in plant transformation range from 15 to 25 kb, but the construction of an artificial binary bacterial chromosome (BIBAC) made the stable insertion of 150 kb possible, which is approximately ten times the previous sequence size (Hamilton et al., 1996). It opens the possibility of the introduction of quantitative traits into other plant genomes. Gelvin (1998) for example, modernly described multigene plant transformation.
Frequently, high levels of expression are desirable, thus constitutive promoters, like 35S from cauliflower mosaic virus (CaMV), have been widely used as single or double copies. Surprisingly, events of partial or complete transgene and/or its endogenous homologue expression inhibition have been observed (Napoli et al., 1990; Van Der Krol et al., 1990). These inactivation events are due to complex mechanisms involving methylation and epigenetic alterations (Renckens et al., 1992; Meyer et al., 1992; Neuhuber et al., 1994; Meyer & Heidmann, 1994; Park et al., 1996; Thierry & Vaucheret, 1996). Although the exact mechanism that targets certain sequences to methyltransferases or other enzymes responsible for epigenetic alterations remains unknown, there are some hypotheses suggesting the involvement of DNA-DNA, DNA-RNA and RNA-RNA homology-dependent interactions (Grierson et al., 1991; Mol et al., 1991; Flavell, 1994; Matzke & Matzke, 1995a; Matzke & Matzke, 1995b; Meyer, 1996; Stam et al., 1997).
Utilization of DNA sequences known as "matrix attachment regions" (MAR) or "scaffold attachment regions" (SAR) has been recommended as a means of keeping chromatin structure accessible to RNA polymerase and the transcription machinery, promoting higher levels and more constant transgene expression (Spiker & William, 1996). Elimination of repeated sequences in transformation vector construction, selection of plants with lower copy numbers and moderated transcription rates in order to favor RNA turnover and the avoidance of multiple copies of the 35S promoter are strategies recommended by Matzke & Matzke (1995b) to avoid (trans)gene silencing. Other promoters able to drive high transcription rates, like chimeric promoters derived from Agrobacterium tumefaciens octopine and manopine synthase genes (Ni et al., 1995), are possible alternatives to maintain high expression levels and reduce homology in order to avoid inactivation phenomena.
Many aspects of plant biology have been elucidated using transgenic plants. There are several reports employing transgenic plants, and a few recent ones are here emphasized: Creté et al. (1997) and Scheible et al. (1997) have elucidated transcription regulation and signal transduction aspects of nitrogen metabolism in higher plants using genetically modified Arabidopsis thaliana, Nicotiana plumbaginifolia and N. tabacum. Molecular identification of plant blue light photoreceptor has also employed transgenic plants (Ahmad & Cashmore, 1993; Lin et al., 1995a; 1995b). Complex biological situations like pathogenesis (Bonas & Van Der Ackerveken, 1997), developmental regulation (Silverstone et al., 1997), genome organization (Murata et al., 1997), reproductive self-incompatibility (Lee et al., 1994) and cross-membrane transportation (Grabov et al., 1997) among many others, have been studied using transgenic plants.
Gene expression and its regulation are frequently studied employing chimeric constructs carrying the promoter fused to a reporter gene, so that induction of the promoter under study is easily assessed by reporter gene expression. However, studies using fusion promoter/reporter genes are being severely criticized (Taylor, 1997), because they do not take into consideration upstream and intragenic sequences that also regulate gene transcription.
Nowadays, many countries have field tests of transgenic plants. As of December 1995, 3,647 genetically engineered plants were field tested worldwide: United States (1952), Canada (486), France (253), United Kingdom (133), Holland (113), Argentina (78), Italy (69), Germany (49), Australia (46), Chile (39), Mexico (38), Spain (30), Japan (25), South Africa (22), Sweden (18), Cuba (18) and Russia (11) among many others. The main transgenic tested field crops are maize, oilseed rape, potato, tomato, soybean, cotton and tobacco, and the most frequent genetically introduced traits are herbicide tolerance (35%), product quality (20%), insect resistance (18%), virus resistance (11%), fungi resistance (3%) and others (13%). The last corresponds to nematode and bacteria resistance and marker or reporter gene transference (Fontes & Carvalho, 1997).
In Brazil, from February to June, 1997, several companies started field tests with transgenic plants. Company names, plant material and the introduced character are listed in Table 1 (Comissão Técnica de Biossegurança - CTNBio, 1997). Birch (1997) shows an extensive list of commercial transgenic plants worldwide. Besides the above mentioned conventional applications, genetically modified plants have also been used in phytoremediation (environmental heavy metal detoxification ) (Rugh et al., 1996), metabolic engineering (Nawrath et al., 1994; John & Keller, 1996) and animal protein production (Ma & Hein, 1995; Rosso & Abad, 1996). Moreover, plant-based vaccines are attractive alternatives to more traditional antigen preparations because of their ease of production and delivery. Arakawa et al. (1998) have developed a transgenic potato that produces human insulin, an insulin-dependent diabetes mellitus autoantigen (IDDM). When the transformed potato was fed to a mouse model of IDDM, the protein was directed to the gut-associated lymphoid tissue, where it induced oral tolerance, as shown by decreased insulitis and suppression of diabetic symptoms.
The main objective of plant transformation is to solve agricultural problems without environmental damage, so that the development of "intelligent plants" able to exclusively kill fungus-infected cells (Strittmatter et al., 1995), or grow taller when there is a shortage of light but grow normally when there are no surrounding plants (Robson et al., 1996), are the goals of modern breeding programs all over the world.
Recebido para publicação em 25.01.99
Aceito para publicação em 25.02.99
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Publication in this collection
18 June 1999
Date of issue
25 Feb 1999
25 Jan 1999