Genetic transformation of the Brazilian BR 451 maize variety by the Agrobacterium tumefaciens method/Transformacao genetica da variedade brasileira de milho BR 451 pelo metodo da Agrobacterium tumefaciens.
Genetic engineering has made new sources of genetic variability available for plant breeding. Great efforts have been made to adapt gene transfer into cereals via A. tumefaciens as they are resistant to such maneuvers (FRAME et al., 2002; 2011). This recalcitrance is related to two different aspects: a) the low in vitro response (CARVALHO et al., 1997; FRAME et al., 2006; GONZALES et al., 2012; GRANDO et al., 2013) e b) low explant sensibility to agrobacteria infection (VENNA et al., 2003; CARVALHO et al., 2004; WANG et al., 2007), thus restricting the number of transformed maize genotypes (GONZALEZ et al., 2012; OMBORI et al., 2014).
The Hi-II maize hybrid and its parents (A188/B73) have been the most frequently used for genetic transformation (ISHIDA et al., 1996; FRAME et al., 2011); however, these genotypes possess inferior agronomic characteristics (ISHIDA et al., 2007). Therefore, it becomes necessary to adapt the A. tumefaciens-mediated transformation parameters to agronomically suitable genotypes, like varieties, hybrids and lines (FRAME et al., 2006; WANG et al., 2007; CARNEIRO et al., 2009; GONZALEZ et al., 2012).
The maize tropical variety BR-451, developed by Embrapa Milho and Sorgo (GUIMARAES et al., 1997), possesses the ability to respond in vitro (CARVALHO et al., 1997). Besides, it exhibits precocity, lodging resistance, high protein content and ability to adapt to many areas in Brazil, which gives it an advantage over the Hi-II hybrid.
Employing genotypes that possess significant agronomic features (like the BR451 maize) and the increased degree of plant regeneration, as well as maintaining infection conditions that encourage the transfer of T-DNA from the agrobacteria to the plant genome (to thus produce a genetic transformation system adapted to alternative genotypes), facilitating the highest degree of genetic engineering and enhancing the quality of this culture in Brazil.
This study aimed to assess the possibility of genetically transforming the Brazilian maize BR 451 through A. tumefaciens, assessing if the high temperatures prevalent when the immature embryos are in the infection phase when h the bacterium raises the transient expression of the UidA reporter gene. This study aimed to confirm whether the growth regulators, when added, enhance in vitro plant regeneration of BR 451 maize variety.
MATERIALS AND METHODS
All the experiments were performed at the Laboratory of Plant Biotechnology at the Universidade de Passo Fundo, using the BR 451 variety of maize genotypes as well as the Hi-II hybrid (functioning as the control).
Regeneration of the BR 451 maize plants
BR 451 mature variety (1-2mm) embryos were cultured in the callus-induction medium according to GRANDO et al., (2013) until the embryogenic calli were formed. Ten subcultures later, the calluses were used for plant regeneration, which involved three stages: (a) germination of the somatic embryos and shoot formation; (b) shoot development and (c) rooting and seedling. One gram of embryogenic callus was distributed in a Petri dish (100mm x 15mm) having 25ml of the different regeneration media to enhance bud formation: M1--basic medium MS plus Erickson vitamins, 100mg [L.sup.-1] of myo-inositol, 60g [L.sup.-1] sucrose, 2g [L.sup.-1] phytagel, pH 5.8; M2--idem to M1 plus 0.5mg [L.sup.-1] of BAP (HUANG & WEI, 2004) and M3--idem to M1, plus 1.5mg [L.sup.-1] of BAP and 0.5mg [L.sup.-1] of IBA (HUANG & WEI, 2005).
All cultures were maintained for one week under dark conditions and then placed under fluorescence lamps of 36.75pmol [m.sup.-2] [s.sup.-1] light intensity to ensure a 16-hour photoperiod in 25 [+ or -] 2[degrees]C. Subculturing was done of the calluses to fresh medium until the shoots emerged, which were then transferred to the MS medium with decreased sucrose concentration (30g [L.sup.-1]) in the absence of growth regulators for the shoots to develop. Ten days later, the shoots were transferred to 300ml vials each with 30ml of half the concentration of MS salts and 30g [L.sup.-1] of sucrose for root and seedling development. Next, the 12cm seedlings were potted (500ml) using equal quantities of MecPlant H01 substrate and carbonized rice husk (1:1) and placed in a humid chamber for 10 days. Finally, they were transferred to a greenhouse under shade cover. Fifteen days later, the plants were planted in to 10L pots until maturity.
The following variables were analyzed: number of shoots, rooted seedlings, plants transferred for acclimatization, acclimatized adult plants and fertile plants per gram of callus. The completely randomized experimental design with five replicates was used, each of which was composed of a Petri dish containing one gram of embryogenic calli. All data were submitted to the analysis of variance.
Temperature stress on embryos infection with A. tumefaciens.
The transformation procedure and culture media employed were based on the protocol of FRAME et al. (2002). The immature embryos (1.2 to 2.0mm) belonging to the BR451 and Hi-II genotypes were placed in the suspension containing the A. tumefaciens strain EHA101: pTF102 for five minutes in the dark, in the infection medium. Embryos at this time were treated at three temperatures: (1) infection at room temperature (25[degrees]C); (2) infection at 40[degrees]C; (3) pretreatment at 40[degrees]C for five seconds with exposure to infection at room temperature.
For three days, the infected explants were co-cultured with A. tumefaciens under conditions of darkness at 20[degrees]C. They were then transferred to the Petri dishes with resting medium for seven days at 28[degrees]C and maintained in the dark. Next, the explants were transferred to the selection medium I supplemented with 1.5mg [L.sup.-1] of Bialaphos[R] and then they were transferred to selection medium II (with 3mg [L.sup.-1] of Bialaphos[R]), continuing to maintain conditions of darkness at 28[degrees]C. Finally, they were subcultured in the same medium every three weeks for six selection cycles until the herbicide resistant callus was formed. A completely randomized experimental design was adopted with 15 replications, where the experimental unit was a Petri dish containing 30 embryos. Transient transformation was evaluated using the GUS histochemical test (JEFFERSON et al., 1987) when the uidA gene (GUS) was expressed three days post infection. The percentage of bluespotted embryos were noted as well as the number of blue spots per GUS positive embryo; five embryos per replicate were used in this evaluation. The frequency of the callus formation resistant to the Bialaphos[R] herbicide was attained only in the sixth selection cycle. Treatments were compared by the means [+ or -] 1 standard deviation.
Verification of the transformation was done by amplifying the bar gene by PCR employing the BarF primers (5'AGAAACCACGTCATGCC3') and BarR (5'TGCACCATCGTCAACCAC3') to react with 25ng of DNA; 2.5nM of each nucleotide; 2.5 [micro]l MgCl; 3[micro]l buffer (5X); 0.15pL Taq polymerase (5U per [micro]L); 10[micro]M of primer 1; 10[micro]M of primer 1 in 25pL of the final volume. The amplification was done by going through one cycle of 94[degrees]C (2min), 35 cycles of 94[degrees]C (20s), 54[degrees]C (20s), 72[degrees]C (30s) and the final extension of 72[degrees]C (4min). Analysis of the amplified product occurred through agarose gel electrophoresis 0.8%; staining was done using ethidium bromide and finally observed in translucent using an ultraviolet lamp (254nm).
RESULTS AND DISCUSSION
Regeneration of BR451 maize plants
Complete and fertile BR 451 plants could be regenerated (Figure 1A). When the BAP and IBA growth regulators were added to the culture medium, regeneration was not affected. An average of 6.4 shoots, 5.4 rooted seedlings and 3.8 plants were subjected to acclimatization and three adult acclimatized plants per gram of callus were produced. These plants were fertile, allowing the transferred characteristics to the offspring, what is important in case of genetic transformation.
Unlike the results observed in this research, HUANG & WEI (2004) verified the effect of 0.5mg [L.sup.-1] BAP on the frequency of shoots of twines. Adopting a similar method, HUANG & WEI (2005) improved the regeneration of maize plants in vitro employing a combination of 1.5mg [L.sup.-1] BAP and 0.5mg [L.sup.-1] IBA. The plant regeneration procedures normally need some alterations for different maize genotypes (CARNEIRO, 2009; FRAME et al., 2011).
In comparison with the other maize genotypes the BR451 regeneration profile reported in this study was satisfactory (three adult plants/ gram of callus). In their experiments CARVALHO et al. (1997) produced between 3.1 and 7.7 plants per gram of callus in 42 tropical lines, including the BR 451 variety, in MS medium, in the absence of growth regulators; although, the authors did not specifically mentioned the frequency of plant regeneration for this variety. The MS medium without the addition of regulators was selected for future regeneration experiments of the BR 451 transgenic plants. This is the same medium used in vitro for the genetic transformation of the responsive maize genotypes (FRAME et al., 2002; 2006).
Effect of temperature on the infection of embryos with A. tumefaciens
When temperature stress was applied at the time of infecting the immature embryos with A. tumefaciens, the frequency of the blue-spotted embryos increased by 50%, as well as the number of blue spots per embryo to 15%, and the percentage of herbicide-resistant callus rose to 29.6% for Hi-II alone (Table 1). The blue dots highlighted the sites where the uidA gene present in the agrobacteria T-DNA was inserted into the maize embryo. This is a measure of transient genetic transformation.
ISHIDA et al., (2007) recorded the positive effect of temperature shock when they achieved 52% transformation using physical stress, plus a temperature of 46[degrees]C for 3min prior to infecting maize embryos. In like manner, pre-treatment at 43[degrees]C (3min) prior to bacterial infection increased the sorghum transformation frequency from 2.6% to 7.6% (GUREL et al., 2009). However, the current experiment indicated that the temperature stress was efficient only when applied during the infection phase. High temperatures were reported to support transformation, most likely by increasing the cell wall permeability (VASCONCELOS et al., 2008). This type of stress could also decrease the degree of the plant tissue defense response to bacterial infection and/or stimulate the protein-controlling genes that assist in activating the vir region.
Apart from the Hi-II hybrid revealing a positive response when exposed to the infection at high temperature, it also showed blue-spotted double embryos (34.3%) compared with the BR 451 (12.7%) variety. Similarly, the number of blue spots per embryo in the Hi-II increased (9.3 points/embryo) and was more than the BR 451 (3.7) showing a higher frequency of Bialaphos[R] resistant callus (22.3%) compared with the BR 451 (5.6%) (Table 1; Figure 1B). The better transformation ability of the Hi-II with agrobacteria has been extensively reported in the literature (ISHIDA et al., 1996; HUANG & WEI, 2005; FRAME et al., 2011).
The difference evident in the response of the genotypes to the transformation is attributed possibly to the variability in the proteins produced, which play a role in the transfer of the agrobacteria T-DNA (ZHANG et al., 2000) and in the plant defense response (VEENA et al., 2003). HWANG & GELVIN (2004) reported that agrobacterial infection induces the plant genes responsible for transformation to be expressed, and at the same time repressed in the host defense response genes.
The presence of the bar gene, which determines the resistance to the Bialaphos[R] herbicide, was identified in the resistant callus via the PCR method. In Figure 1C the amplification of the bar gene in certain calli of the Hi-II and BR451 genotypes is evident. However, the bar gene was missing in some resistant calli, implying the presence of scapes, which entails adjustment of the selection pressure to the selective process. This study is the first report of the genetic transformation of the BR 451 maize genotype via A. tumefaciens.
The maize BR 451 variety was amenable to genetic transformation utilizing A. tumefaciens, implying the likelihood of its use in generating genetically modified plants with significant agronomic genes. When the immature embryos of this variety were infected by the agrobacteria, the application of temperature stress does not affect the transient transformation of the reporter gene. Similarly, when growth regulators were added to the culture medium the regeneration of the BR 451 plants was not encouraged.
Received 08.22.14 Approved 07.04.17 Returned by the author 09.04.17
We are grateful to Dra. Andreia Almeira Carneiro from Embrapa Maize and Sorghum for the concession of the BR 451 maize variety.
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Marilia Rodrigues de Silva (1) Dielli Aparecida Didone (1) Cassia Canzi Ceccon (1) Vinicius de Oliveira Almeida (1) Magali Ferrari Grando (1) *
(1) Laboratorio de Biotecnologia Vegetal, Faculdade de Agronomia e Medicina Veterinaria, Universidade de Passo Fundo (UPF), 99052-900, Passo Fundo, RS, Brasil. E-mail: email@example.com. * Corresponding author.
Caption: Figure 1--Regeneration of plants in vitro and genetic transformation of the maize BR 451 variety. A) In vitro regeneration process. a, b) bud formation from embryogenic calli cultivated in M1 regeneration medium; c) maize seedlings in flasks containing lA MS; d) plant acclimatized in pot with substrate; e) potted plants in greenhouses; f) Ear produced by plant regenerated in vitro in M1 medium. B) Expression of the uidA gene (blue spots) after GUS histochemical assay on immature embryos submitted to different temperature treatments in the infection stage with A. tumefaciens and callus resistant to the Bialaphos[R] herbicide of the Hi-II and BR 451 genotypes after six cycles of selection. C) PCR analysis for the bar gene in callus resistant to the herbicide Bialaphos[R]. M: 1000pb marker; C+: isolated plasmid DNA from the A. tumefaciens EHA101: pTF102 as a positive control; C-: untransformed callus as negative control; B: Blank; 1: negative callus for bar gene; 2,3,4: embryogenic calli containing the bar gene presenting a PCR amplified 407bp fragment, Passo Fundo, FAMV/UPF, 2014.
Table 1--Expression of the uidA gene and the production of Bialaphos[R] herbicide resistant calli in two maize genotypes submitted to different treatments in the stage of infection of immature embryos with Agrobacterium tumefaciens EHA101/pTF102. Passo Fundo, FAMV/UPF, 2012. Genotype Treatment Percentage of Number of Percentage of embryos with blue spots Bialaphos[R] blue spots per embryo resistant callus Hi-II infection at 31 7 18.2 25[degrees]C Hi-II infection at 50 S 15 S 29.6 S 40[degrees]C Hi-II pretreatment 22 5.4 19.1 at 40[degrees]C followed by infection at 25[degrees]C. Mean 34.3 9.3 22.3 BR 451 infection at 13.6 1.6 7.4 25[degrees]C BR 451 infection at 11.7 1.1 1.1 40[degrees]C BR 451 pretreatment 12.6 8.5 8.5 at 40[degrees]C followed by infection at 25[degrees]C. Mean 12.7 3.7 5.6 M + 1dp 43.4 10.7 24.5 M - 1dp -9.7 -3.7 -4.6 S = superior (Mean plus one standard deviation).