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Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

Abstract

Low transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciens overgrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future.

Keywords:
co-cultivation conditions; protocol establishment; GUS; T-DNA delivery

Wheat (Triticum aestivum) is one of the most cultivated cereal crops worldwide and is a major source of protein and carbohydrates to humans. Nevertheless, like all the crops, it is faced with various biotic and abiotic diseases and other important challenges such as special quality characteristics and improved productivity. These traits can be obtained with the aid of transgenesis (James, 2013James C (2013). Global Status of Commercialized Biotech/GM Crops: 2013. ISAAA Brief No. 46. ISAAA, Ithaca.).

Several improvements have been made in the wheat transformation protocol since transgenic wheat was first created using Agrobacterium tumefaciens in 1997 (Cheng et al., 1997Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW and Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971-980.). However, despite a range of protocols that have been developed since then (Cheng et al., 2003Cheng M, Hu TC, Layton J, Liu CN and Fry JE (2003) Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cell Dev Biol Plant 39:595-604.,2004Cheng M, Lowe BA, Spencer TM, Ye XD and Armstrong CL (2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev Biol Plant 40:31-45.; Hu et al., 2003Hu T, Metz S, Chay C, Zhou HP, Biest N, Chen G, Cheng M, Feng X, Radionenko M, Lu F, et al. (2003) Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep 21:1010-1019.; Khanna and Daggard, 2003Khanna HK and Daggard GE (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Rep 21:429-436.; Wu et al., 2003Wu H, Sparks C, Amoah B and Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21:659-668.; Jones et al., 2005Jones HD (2005) Wheat transformation: Current technology and applications to grain development and composition. J Cereal Sci 41:137-147.; Patnaik et al., 2006Patnaik D, Vishnudasan D and Khurana P (2006) Agrobacterium-mediated transformation of mature embryos of Triticum aestivum and Triticum durum. Curr Sci India 91:307-317.; Agarwal et al., 2009Agarwal S, Loar S, Steber C and Zale J (2009) Floral transformation of wheat. In: Jones HD and Shewry PR (eds) Transgenic Wheat, Barley and Oats. Series Methods in Molecular Biology. Humana Press, New Jersey, 478:105-113.; Ding et al., 2009Ding L, Li S, Gao J, Wang Y, Yang G and He G (2009) Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Mol Biol Rep 36:29-36.; Risacher et al., 2009Risacher T, Craze M, Bowden S, Paul W and Barsby T (2009) Highly efficient Agrobacterium-mediated transformation of wheat via in planta inoculation. In: Jones HD and Shewry PR (eds) Transgenic Wheat, Barley and Oats. Series Methods in Molecular Biology. Humana Press, New Jersey, 478:115-124.), progress in hexaploid T. aestivum has been slower than in tetraploid species (Wu et al., 2008Wu H, Doherty A and Jones HD (2008) Efficient and rapid Agrobacterium-mediated genetic transformation of durum wheat (Triticum turgidum L. var. Durum) using additional virulence genes. Transgenic Res 17:425-436.; He et al., 2010He Y, Jones HD, Chen S, Chen XM, Wang DW, Li KX, Wang DS and Xia LQ (2010) Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency. J Exp Bot 61:1567-1581.) and other cereals such as maize (Vega et al., 2008Vega JM, Yu W, Kennon AR, Chen X and Zhang ZJ (2008) Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep 27:297-305.) and rice (Karthikeyan et al., 2011Karthikeyan A, Pandian SK and Ramesh M (2011) Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43). Plant Sci 181:258-268.). Hiei et al. (2014Hiei Y, Ishida Y and Komari T (2014) Progress of cereal transformation technology mediated by Agrobacterium tumefaciens. Front Plant Sci 5:e628.) stated that the optimal ranges of many of the factors are narrow in wheat, and that this can be a reason for the slow progress made. The only known protocol giving high stable transformation efficiency is from Japan Tobacco (Ishida et al., 2015Ishida Y, Tsunashima M, Hiei Y and Komari T (2015) Wheat (Triticum aestivum L.) transformation using immature embryos. In: Wang K (ed) Agrobacterium Protocols: Vol. 1. Series Methods Mol Biol. Springer+Business Media, New York, 1223:189-198.), for which use is permitted under license payment.

This difficulty encountered for wheat is due to its huge genome that contains large amounts of repetitive DNA, as well as its low regeneration capacity and resistance to A. tumefaciens infection (Bhalla, 2006Bhalla PL (2006) Genetic engineering of wheat - Current challenges and opportunities. Trends Biotechnol 24:305-311.). Only few studies have analyzed concomitantly the factors related to increased infectivity, such as pH, temperature and acetosyringone (AS) concentration (Gelvin, 2003Gelvin SB (2003) Agrobacterium-mediated plant transformation: The biology behind the “Gene-jockeying” tool. Microbiol Mol Biol Rev 67:16-37.). It is known that an acid environment and AS act directly on the expression of the A. tumefaciens vir region (McCullen and Binns, 2006McCullen AC and Binns AN (2006) Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol 22:101-127.). Furthermore, temperature is associated with T-pilus formation (Lai et al., 2000Lai EM, Chesnokova O, Banta L and Kado CI (2000) Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens. J Bacteriol 182:3705-3716.). It has been suggested that these factors act together in the beginning of the transformation processes inducing the expression of vir genes (He et al., 2010He Y, Jones HD, Chen S, Chen XM, Wang DW, Li KX, Wang DS and Xia LQ (2010) Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency. J Exp Bot 61:1567-1581.). So far, nothing is known about the liability of Brazilian genotypes for genetic transformation mediated by A. tumefaciens, but there are great interests in using genetic material presenting good agronomic traits.

For the establishment of a genetic transformation protocol two main things are: improval of the conditions for T-DNA delivery and the regeneration response of the plant cells. Therefore, we attempted to adjust the factors responsible for the induction of A. tumefaciens virulence by determining the best combinations of pH, temperature and phenolic compound (AS) for T-DNA delivery into wheat genomes, as well as its effect on the regeneration of immature embryos subjected to the transformation processes. Two Brazilian wheat genotypes having good agronomic traits were compared with respect to above-mentioned variables. To our knowledge, this work is the first to evaluate concomitantly these main factors related to the early A. tumefaciens infection process in wheat.

Donor plants were the Brazilian wheat genotypes PF 020037 and BR 18 Terena. They were grown in 8 L pots in growth chambers with a photoperiod of 16/8 h, a temperature cycle of 21 °C/14 °C (day/night) and light intensity of approximately 150 μmol.m-2.s-1. Ears were marked at anthesis and harvested after 11 to 14 days, when immature embryos were 0.8 to 1.5 mm long, cream-colored and translucent in appearance. Seeds were disinfested with 70% ethanol (v/v) for 1 min followed by a solution of commercial bleach (2.0 to 2.5% active chlorine) diluted to 1:1 (v/v) in water containing 0.1% (v/v) Tween 20 for 10 min, followed by five rinses with sterile distilled water. Immature embryos were collected aseptically, embryo axes were removed and then placed scutellum-side up in a Petri dish containing co-cultivation medium (Table S1 Table S1 - Culture medium composition ).

A. tumefaciens strain AGL1 (Lazo et al., 1991Lazo GR, Stein PA and Ludwig RA (1991) A DNA transformation competent Arabidopsis genomic library in Agrobacterium. Bio-Technology 9:963-967.) containing the plasmids pAL154 and pAL156, which is based on pSoup/pGreen vectors (Hellens et al., 2000Hellens RP, Edwards EA, Leyland NR, Bean S and Mullineaux PM (2000) pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819-832.), was provided by Dr. Huw Jones, Rothamsted Research, UK. The helper plasmid pAL154 contained the 15 kb Komari fragment (Komari, 1990Komari T (1990) Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Rep 9:303-306.) that provides replication functions for pAL156 in trans. The Komari fragment possesses additional vir genes (virG542, virB e virC). pAL156 T-DNA contains the bar gene (conferring resistance to glufosinate ammonium) and a modified reporter gene, uidA gene + intron. Both genes are driven by the Ubi+Intron (maize ubiquitin plus an intron) promoter and the nopaline synthase terminator (nos) (Figure S1 Figure S1 - Schematic map of the T-DNA region of the pAL156 vector ).

Genetic transformation was performed according to Wu et al. (2009Wu H, Doherty A and Jones HD (2009) Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos. In: Jones HD and Shewry PR (eds) Transgenic Wheat, Barley and Oats. Series Methods in Molecular Biology. Humana Press, New Jersey, 478:93-103.), with modifications for the treatments analyzed. Bacteria from glycerol stocks were grown on solid LB medium (10 g L-1 Bacto tryptone, 5 g L-1 Bacto yeast extract, 5 g L-1 NaCl and 15 g L-1 agar) supplemented with 100 mg L-1 carbenicillin, 50 mg L-1 rifampicin and 50 mg L-1 kanamycin. One or two streaks of bacterial cells harvested with an inoculation loop were grown in 10 mL of liquid LB medium containing the same antibiotics for 17 to 20 h at 28 °C and 250 rpm. Bacterial cell suspensions were centrifuged at 4,500 x g for 10 min. The supernatant was discarded, the pellet resuspended in inoculation medium (Table S1 Table S1 - Culture medium composition ), and the cell concentration was adjusted to an OD600 of 1.0 to 2.0. This suspension was supplemented with AS and incubated at 200 rpm and 22 °C for one to three hours. Plates containing approximately 50 embryos received 3 mL of the bacterial suspension supplemented with 0.015% Silwet L77 and were maintained in the dark for 15 min. After removing the bacterial suspension the embryos were transferred to fresh co-cultivation medium and maintained in the dark for three days. Subsequently, the embryos were transferred to induction medium (Table S1 Table S1 - Culture medium composition ). After 3-4 weeks in the dark, the calli were transferred to regeneration medium (Table S1 Table S1 - Culture medium composition ) and grown for an additional three weeks under 30 μmol.m-2.s-1 light intensity and 16 hours photoperiod. Calli with plantlets were subjected to two rounds of incubation for 3-4 weeks in selective medium (Selection 1 and 2, Table S1 Table S1 - Culture medium composition ).

Expression of the uidA gene in transgenic tissues was detected by histochemical test for β-glucuronidase (GUS) (Jefferson et al., 1987Jefferson RA, Kavanagh TA and Bevan MW (1987) GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in high plants. EMBO J 6:3901-3907.). Explants were sampled after 2-3 days on induction medium and incubated at 37 °C for 16 h in X-Gluc buffer [1 mM X-Gluc, 100 mM sodium phosphate buffer pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% (v/v) Triton X-100 with 20% methanol (v/v)]. After this step, the embryos were rinsed with 70% ethanol and stored in the same solution at 4 °C until blue foci were counted. As the counting of GUS foci is very subjective, the same person assessed all experiments, in an attempt to avoid misinterpretations.

Initially, we evaluated the effects of pH, temperature and AS concentration on T-DNA delivery and regeneration of plantlets after infection of immature embryos of the wheat line PF 020037. The factorial experiment (3×3×2) had three pH (5.0, 5.4 and 5.8), three co-cultivation temperature (19, 22 and 25 °C) and two AS concentration levels (200 and 400 μM), and were evaluated using a completely randomized design. Each treatment consisted of approximately 140 immature embryos, with 35 being control embryos (not inoculated) and the remainder inoculated with A. tumefaciens. Fifty inoculated embryos were used for histochemical tests, and the remainder proceeded to the subsequent stages of the evaluation protocol. The percentage of embryos with at least one blue focus (% of GUS+) and the number of blue foci per embryo were recorded, and the regeneration frequency (percentage of immature embryos that produced shoots) was determined. This experiment was repeated once. The three best combinations obtained for the wheat line PF 020037 were then used to assess the response of the wheat cultivar BR 18 Terena to T-DNA delivery. The percentage data were transformed by, csinx100 and counting data were transformed by (x)+(x+1). Data were subjected to ANOVA for F tests, and means were compared by the Tukey post-hoc test. At p < 0.05 differences were considered as statistically significant.

The three factors studied showed significant interaction (p < 0.05) for both the percentage of GUS+ embryos and the number of blue foci per embryo. Combinations more favorable to infection were seen for media with pH values of 5.0 and 5.4, co-cultivation temperatures of 22 and 25 °C, and, preferably, with the addition of 400 μM AS (Figure 1A-C). Among the media adjusted to a pH of 5.0, the most favorable combinations occurred at 25 °C with 400 μM AS (81.4% GUS+ embryos and 5.7 blue foci per embryo) and at 22 °C with 200 μM AS (71.6% GUS+ embryos and 4.0 blue foci per embryo) (Table 1).

Figure 1
Transient uidA (GUS) gene expression in immature embryos of wheat genotype PF 020037 inoculated with Agrobacterium tumefaciens. Combinations more favorable for T-DNA expression were: A) pH 5.0 – 22 °C – 200 μM AS; B) pH 5.0 – 25 °C – 400 μM AS;. C) pH 5.4 – 22 °C −400 μM AS. Combinations less favorable for T-DNA expression were: D) pH 5.8 – 19 °C −200 μM AS; E) pH 5.8 – 22 °C −200 μM AS.; F) pH 5.8 – 25 °C – 400 μM AS.
Table 1
Effect of the interaction between pH, temperature and acetosyringone (AS) concentration on T-DNA delivery.

When the inoculation and co-cultivation media were adjusted to a pH of 5.4, the highest rate of T-DNA delivery was obtained at 22 °C with 400 μM AS (77.5% GUS+ embryos and 4.6 blue foci per embryo). Nonetheless, these means were not significantly different from the ones obtained at 25 °C with 400 μM AS (55.9% GUS+ embryos and 2.8 blue foci per embryo). Culture media adjusted to pH 5.8 did not provide favorable combinations for infection (Figure 1D-F) and yielded lower infection frequencies than pH values of 5.0 and 5.4 for all combinations of temperature and AS concentration, with the exception of 25 °C and 200 μM AS.

Considering the four treatments that showed significant differences between the two AS concentrations investigated, culture media containing 400 μM resulted in higher T-DNA delivery at three different pH and temperature combinations (pH 5.0 −25 °C; pH 5.4 −22 °C and pH 5.4 −25 °C). The best treatment when using 200 μM AS was in a combination of pH of 5.0 with 22 °C. Co-cultivation temperatures of 22 and 25 °C were more favorable for infection, except for combinations of pH 5.4 and 200 μM AS, and for pH 5.8 and 400 μM AS. These two combinations showed equal means at the three temperatures investigated (Table 1).

The two most recent protocols relevant for wheat transformation used 400 μM (Sparks et al., 2014Sparks CA, Doherty A and Jones H (2014) Genetic transformation of wheat via Agrobacterium-mediated DNA delivery. In: Henry RJ and Furtado A (eds) Cereal Genomics: Methods and Protocols. Series Methods in Molecular Biology. Springer+Bisiness Media, New York, 1099:235-250.) and 100 μM AS (Ishida et al., 2015Ishida Y, Tsunashima M, Hiei Y and Komari T (2015) Wheat (Triticum aestivum L.) transformation using immature embryos. In: Wang K (ed) Agrobacterium Protocols: Vol. 1. Series Methods Mol Biol. Springer+Business Media, New York, 1223:189-198.), respectively, both using Fielder. Sparks et al. (2014Sparks CA, Doherty A and Jones H (2014) Genetic transformation of wheat via Agrobacterium-mediated DNA delivery. In: Henry RJ and Furtado A (eds) Cereal Genomics: Methods and Protocols. Series Methods in Molecular Biology. Springer+Bisiness Media, New York, 1099:235-250.) suggest the use of 200 μM to 800 μM AS. Thus, even though the AS concentration is important, it does not seem to be the main factor that needs to be optimized, as the most efficient protocol published by Ishida et al. (2015Ishida Y, Tsunashima M, Hiei Y and Komari T (2015) Wheat (Triticum aestivum L.) transformation using immature embryos. In: Wang K (ed) Agrobacterium Protocols: Vol. 1. Series Methods Mol Biol. Springer+Business Media, New York, 1223:189-198.) uses only 100 μM.

The overgrowth of A. tumefaciens was more pronounced when pH and temperature were increased (Table 2). The regeneration frequency of control explants was greater than for the inoculated ones for all combinations, and it remained stable regardless of pH. Therefore, the immature embryos were probably damaged by A. tumefaciens overgrowth during co-cultivation and not by the culture media condition itself. Among all combinations analyzed, regeneration frequencies ranged from 23.1 to 58.1% for the control and from 0 to 31% for the inoculated explants. A. tumefaciens overgrowth in media with a pH 5.8 correlated with reduced explant regeneration capacity. The average loss of capacity to regenerate in these media was of 89.1%, which is far higher than the 24.9% average loss in media with a pH of 5.0 (data not shown). No relevant losses were observed when comparing the two AS concentrations. The transient transformation increased when using a 400 μM AS concentration at pH 5.4, but explant regeneration capacity decreased. Similar behavior was, however, not observed in media with a pH of 5.0. Although some plants survived the selection step, no transgenic plants were confirmed by PCR targeting the bar and uidA genes. We suspect that the phosphinothricin (PPT) concentration used in the selection medium was not sufficient to prevent the survival of non-transgenic plants. Increasing this concentration to 10 mg L-1, as used by Ishida et al. (2015Ishida Y, Tsunashima M, Hiei Y and Komari T (2015) Wheat (Triticum aestivum L.) transformation using immature embryos. In: Wang K (ed) Agrobacterium Protocols: Vol. 1. Series Methods Mol Biol. Springer+Business Media, New York, 1223:189-198.), may reduce this problem. Also, it was observed that A. tumefaciens prefers to infect scutellar borders, which are considered less likely for callus initiation (Ke et al., 2002Ke XY, McCormac AL, Harvey A, Lonsdale D, Chen DF and Elliott MC (2002) Manipulation of discriminatory T-DNA delivery by Agrobacterium into cells of immature embryos of barley and wheat. Euphytica 126:333-343.; McCormac et al., 1998McCormac AC, Wu H, Bao M, Wang Y, Xu R, Elliot MC and Chen DF (1998) The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). Euphytica 99:17-25.) than wound regions, which could be another reason for not obtaining transgenic plants.

Table 2
Regeneration frequency of wheat immature embryos (PF 020037 genotype) not inoculated (control) and inoculated with A. tumefaciens, as well as bacteria overgrowth levels during co-cultivation.

The three best combinations found in the first experiment [(I) pH 5.0 – 25 °C – 400 μM AS, (II) pH 5.4 −22 °C – 400 μM AS e (III) pH 5.0 −22 °C – 200 μM AS] were the treatments used to compare the response to GUS expression in BR 18 Terena and PF 020037. There was no interaction between combinations (I, II, and III) regarding genotypes (p > 0.05) and there was no significant difference between them for the percentage of GUS+ embryos and the number of blue foci per embryo (Table 3). However, BR 18 Terena was superior, as the percentage of GUS+ embryos of 71.8% as 13% higher than the one observed in PF 020037 (58.8%). For the number of blue foci per embryo the two wheat genotypes were similar to GUS+ response (5.2 in BR 18 Terena and 2.8 in PF 020037).

Table 3
Effect of wheat genotype on T-DNA delivery.

The basic requirement for a successful gene transfer system is the ability to transfer an intact DNA segment into the nuclear genome of regenerable cells and the ability to recover adult plants from transformed cells via tissue culture (Jones, 2005Jones HD, Doherty A and Wu H (2005) Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 1:5.). Our work shows that it is possible to improve T-DNA delivery into plant cell genomes by adjusting the factors responsible for inducing the vir region, such as pH, temperature and AS concentration (McCullen and Binns, 2006McCullen AC and Binns AN (2006) Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol 22:101-127.).

Our results (Table 1, Figure 1) support the hypothesis that AS interacts with specific temperatures and acidic environments to promote the expression of vir genes in A. tumefaciens during the inoculation and co-cultivation period (He et al., 2010He Y, Jones HD, Chen S, Chen XM, Wang DW, Li KX, Wang DS and Xia LQ (2010) Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency. J Exp Bot 61:1567-1581.). The majority of published wheat genetic transformation protocols report pH values of 5.7 – 5.8 for the culture medium (Cheng et al., 2004Cheng M, Lowe BA, Spencer TM, Ye XD and Armstrong CL (2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev Biol Plant 40:31-45.; Jones et al., 2005Jones HD (2005) Wheat transformation: Current technology and applications to grain development and composition. J Cereal Sci 41:137-147.; Wu et al., 2009Wu H, Doherty A and Jones HD (2009) Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos. In: Jones HD and Shewry PR (eds) Transgenic Wheat, Barley and Oats. Series Methods in Molecular Biology. Humana Press, New Jersey, 478:93-103.). However, more acidic pHs (5.2-5.4) are described in the transformation protocols of other monocots such as maize (Frame et al., 2006Frame BR, McMurray JM, Fonger TM, Main ML, Taylor KW, Torney FJ, Paz MM and Wang K (2006) Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts. Plant Cell Rep 25:1024-1034.; Vega et al., 2008Vega JM, Yu W, Kennon AR, Chen X and Zhang ZJ (2008) Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep 27:297-305.) and rice (Hiei and Komari, 2006Hiei Y and Komari T (2006) Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 85:271-283.; Patel et al., 2013Patel M, Dewey RE and Qu R (2013) Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection. Plant Cell Tissue Organ Cult 114:114-129.). At pH 5.0, a strong trend towards increased T-DNA delivery with increasing AS concentration was observed without any negative effects on the regeneration potential of immature embryos. However, at pH 5.4, regeneration capacity was affected when T-DNA delivery was increased by increasing the AS concentration. He et al. (2010He Y, Jones HD, Chen S, Chen XM, Wang DW, Li KX, Wang DS and Xia LQ (2010) Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency. J Exp Bot 61:1567-1581.) reported that increasing transient transformation by increasing AS concentration from 200 to 400 μM did not decrease the regeneration frequency of somatic embryos; however, this work was carried out using T. durum and at pH of 5.8. As bacterial overgrowth occurs in treatments utilizing culture media with pH 5.4, embryo tissues have probably been sufficiently damaged to compromise the regeneration but not the transformation and expression of the T-DNA. This is an example for the claim by Hiei et al. (2014Hiei Y, Ishida Y and Komari T (2014) Progress of cereal transformation technology mediated by Agrobacterium tumefaciens. Front Plant Sci 5:e628.), that for wheat the optimal ranges of many of the factors for transformability are narrow.

In this study, we observed, by visual analysis, that the occurrence of overgrowth by A. tumefaciens is less during co-cultivation in culture media with pH 5.0 than with those of pH 5.4 and 5.8. Fewer bacteria likely represent reduced damage to embryo tissues, resulting in better plantlet regeneration. Wu et al. (2009Wu H, Doherty A and Jones HD (2009) Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos. In: Jones HD and Shewry PR (eds) Transgenic Wheat, Barley and Oats. Series Methods in Molecular Biology. Humana Press, New Jersey, 478:93-103.) reported that A. tumefaciens overgrowth during co-cultivation is a problem for the regeneration of immature embryo calluses when using the same strain and inoculation and similar co-cultivation medium pH (5.7). An alternative to decreasing A. tumefaciens overgrowth during co-culture may be to reduce co-cultivation time from three to two days. However, care must be taken, as this reduction may affect T-DNA expression. To overcome the overgrowth problem Khanna and Daggard (2003Khanna HK and Daggard GE (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Rep 21:429-436.) decreased the temperature after the first day of co-culture. These authors also included spermidine in the induction medium to facilitate the recuperation of infected tissues. Lowering the bacterial concentration or rinsing the explants with liquid culture medium, has also been recommended (Hensel et al., 2009Hensel G, Kastner C, Oleszcuk S, Riechen J and Kumlehn J (2009) Agrobacterium-mediated gene transfer to cereal crop plants: Current protocols for barley, wheat, triticale and maize. Int J Plant Genomics 2009:e835608.).

The Brazilian wheat cultivar BR 18 Terena demonstrated good potential for genetic transformation, as it is more susceptible to A. tumefaciens than PF 020037. Both genotypes were used because they showed ability to regenerate and capability of transient transformation in previous experiment. In addition, under the same assay conditions, the model genotype Bobwhite had very low levels of transient transformation. This finding probably correlates with the higher adaptability of the Brazilian genotypes to the growth conditions of the donor plants. The fact that BR 18 Terena is an elite wheat cultivar should facilitate the introduction of transgenes into breeding programs. In conclusion, the joint analysis of key factors, such as pH, temperature and AS, involved in the early stages of A. tumefaciens-mediated transformation revealed that such factors are not simply additive and can be manipulated to improve the efficiency of T-DNA delivery and to control bacterial overgrowth during co-cultivation.

Acknowledgments

This study was funded by the Brazilian Agricultural Research Corporation (EMBRAPA), Coordination for the Improvement of Higher Education Personnel (CAPES) and National Council for Scientific and Technological Development (CNPq). We are grateful to Dr. Huw Jones and Ms. Caroline Sparks (Rothamsted Research, UK) for providing Agrobacterium tumefaciens AGL1 pAL154/pAL156 and useful suggestions. We also thank Andréa Morás and Lucimere F. Morelo for their skillful technical assistance, Márcio Nicolau and Ricardo L. Castro for support with statistical analysis, and Fátima M. Marchi for the artwork.

  • Associate Editor: Dario Grattapaglia

Supplementary Material

The following online material is available for this article:

Figure S1 - Schematic map of the T-DNA region of the pAL156 vector

Table S1 - Culture medium composition

This material is available as part of the online article from: http://www.scielo.br/gmb.

References

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Publication Dates

  • Publication in this collection
    03 Nov 2015
  • Date of issue
    Oct-Dec 2015

History

  • Received
    23 Jan 2015
  • Accepted
    07 July 2015
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