Stable and efficient Agrobacterium tumefaciens mediated transformation of Phaseolus vulgaris

Transformación estable y eficiente de Phaseolus vulgaris mediada por Agrobacterium tumefaciens

Elsa Espinosa-Huerta, Anareli Quintero-Jiménez¹, K. Virginia Cabrera-Becerra, M. Alejandra Mora-Avilés*

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Unidad de Biotecnología de Plantas. Km. 6.5 Carretera Celaya-San Miguel de Allende, Apartado Postal 112, Celaya, 38110 Guanajuato, México. * Author for correspondence. (mora.alejandra@inifap.gob.mx).

Received: August, 2012.
Approved: April, 2013.

Abstract

Common bean breeding requires novel techniques to incorporate genes that may not be available in bean genetic diversity. The objective of the present study was to analyze the main variables involved in Agrobacterium tumefaciens-mediated transformation of common bean towards providing an efficient system to introduce new agronomic traits. Bean cultivars Flor de Mayo Anita and Pinto Saltillo were inoculated with different A. tumefaciens strains, constructs and selection was done using two selection agents (kanamycin ot glufosinateammonium herbicide). Five-day old hypocotyls regenerated organogenic buds (cell clusters) 5 d after inoculation. A randomized complete blocks experimental design was used and the experimental unit was a petri dish with at least 10 hypocotyls. Analysis of variance and Tukey test (p≤0.05) were perfotmed on data. Transformation efficiency was 10-28 % with small variations due to the genetic backgtound of each cultivar, the Agrobacterium strain and selection agent used. Transfotmation efficiency was higher using kanamycin (28.6 %) in relation to glufosinate-ammonium (10.2 %). Similarly, Pinto Saltillo cultivar showed better regeneration response (21-28 %) than that of Flor de Mayo Anita cultivar (10-17 %) and this was consistent when selection agents were compared within each cultivar. Molecular analysis for detection and gene expression quantification by using end point PCR and q-PCR showed evidence of presence and activity at different expression levels through several generations (T0 to T3). transformed lines of common bean with potential for fungal resistance and drought tolerance were successfully obtained via Agrobacterium tumefaciens with evidence of generic stability.

Key words: bud clusters, drought stress tolerance, fungal resistance, Gamborg medium, hypocotyls, organogenesis.

Resumen

El mejoramiento del frijol común requiere técnicas novedosas para incorporar genes que puedan no estar disponibles en la diversidad genética del frijol. El objetivo del presente estudio fue analizar las principales variables involucradas en la transformación mediante Agrobacterium tumefaciens de frijol con el propósito de proveer un sistema eficiente pata introducir nuevas catactetísticas agronómicas. Los cultivares de frijol Flor de Mayo Anita y Pinto Saltillo fueron inoculados con diferentes cepas de A. tumefaciens y construcciones. La selección se realizó usando dos agentes, la kanamicina o el herbicida glufosinato de amonio. Hipocótilos de 5 d regeneraron brotes organogénicos (grupos de células) 5 d después de la inoculación. El diseño experimental fue bloques completos al azar y la unidad experimental fue una placa de petri con al menos 10 hipocótilos. Un análisis de varianza y la prueba de Tukey (p≤0.05) se efectuaton con los datos. La eficiencia de transformación fue 10-28 %, con variaciones pequeñas debido a los antecedentes genéticos de cada cultivar, la cepa de Agrobacterium y el agente de selección usado. La transformación fue más eficiente usando kanamicina (28.6 %) en relación al glufosinato de amonio (10.2 %). Similarmente, el cv. Pinto Saltillo mostró mejor respuesta de regeneración (21-28 %) que Flor de Mayo Anita (10-17 %) y esto fue consistente al comparar los agentes de selección dentro de cada cultivar. El análisis molecular para la detección y cuantificación de la expresión génica mediante PCR punto final y q-PCR mostró evidencia de su presencia y actividad en los diferentes niveles de expresión a través de varias generaciones (T0 a T3). Líneas de frijol transformadas con potencial para resistir hongos patógenos y toletancia a sequía se obtuvieron exitosamente mediante Agrobacterium tumefaciens, con evidencia de estabilidad genética.

INTRODUCTION

Common bean (Phaseolus vulgaris L.) is a crop with vast genetic diversity that includes germplasm with superior agronomic traits such as resistance to adverse weather conditions, pest and disease resistance, and high nutritional quality (Cruz de Carvalho et al., 2000). However, some genes that confer specific traits are either not present in common bean germplasm or conventional breeding may result time consuming. Most breeding programs have made significant progress in the development of disease resistant or drought tolerant cultivars; nevertheless, there is need for the incorporation of more efficient genes with low metabolic cost for the plant.

A protocol that provides efficient cell differentiation, shoot development, and whole plant regeneration is an essential requirement for genetic transformation in plants. Originally, only three groups had produced stable and heritable transformation of P. vulgaris (Russell et al., 1993; Aragão et al., 1998) and P. acutifolius (Dillen et al., 1997). However, there are successful transformation events including the utilization of Agrobacterium rhizogenes to generate transformed roots, research oriented towards functional genomics (Estrada-Navarrete et al., 2006), as well as transformation via particle bombardment to develop bean cultivars resistant to the bean golden yellow mosaic virus through the insertion of a mutated rep gen (Faria et al., 2006; Bonfim et al., 2007).

Direct organogenic regeneration of common bean for two commercial cultivars with high regeneration efficiency was reported by Delgado-Sánchez et al. (2006) and Quintero-Jiménez et al. (2010a; 2010b). Some factors need to be taken into consideration for a consistent regeneration of transformed plants. Several types of explants give differential response, as cotyledons (McClean et al., 1991), protoplasts (Leon et al., 1991), mature seed embryos (Aragão et al., 1992), leaf disks and hypocotyls (Franklin et al., 1993), meristems (Russell et al., 1993; Brasileiro et al., 1996), apical shoots (Lewis and Bliss, 1994) and embryonic axes (Kim and Minamikawa, 1996) . Seed age, seed size and color as well as culture media are traits to consider. Transformation via particle bombardment was reported with effective results (Russell et al., 1993; Aragão et al, 1996; 1998; 2002; Rech et al., 2008). Moreover, Agrobacterium strain can be a qualitative determinant factor for success in the development of common bean lines genetically modified (McClean et al., 1991; Zambre et al., 1998; 2005).

The objective of the present study was to analyze variables involved into a stable and efficient Agrobacterium mediated transformation of common bean by introducing two genes of agronomic importance.

MATERIALS AND METHDOS

Plant material

Constructs

Defensine (pdf1.2) and proton pump pyrophosphatase (avpJ) genes confer resistance to fungal pathogens (Raharjo et al., 2008) and drought tolerance (Gaxiola et al., 2001; Park et al, 2005). Thepdf1.2 cDNA gene (0.439 kb) from Arabidopsis thaliana (Penninckx et al., 1996) was inserted into the pKYLX80 binary vector (Schardl et al., 1987). The pdf1.2 gene was regulated by a double cauliflower mosaic virus (CaMV) 35S promoter and the small subunit polypeptide rubisco terminator from pea (rbcS-E9). The neomycin phosphotransferase gene (nptII) provides kanamycin resistance under control of the nopaline synthase (nos) promoter and nos terminator from A. tumefaciens (Figure 1A). The plasmid was inserted in Agrobacterium strain GV2260 by heat shock transformation (Hoisington et al., 1994). The avp1 open reading frame (2313 bp) from A. thaliana was cloned into the XbaI site of a modified pRT103 plasmid (Gaxiola et al., 2001). The avp1 gene was regulated by a double cauliflower mosaic virus (CaMV) 35S promoter and A. tumefaciens nos terminator. The phosphinothricin acetyl transferase gene (pat/ bar) from Streptomyces hygroscopicus which confers resistance to glufosinate-ammonium herbicide, under control of nos promoter and nos terminator, acted as selectable marker (Figure 1B). The plasmid was inserted into A. tumefaciens strain GV3101.

Transformation procedure

Seed germination, hypocotyls dissection and media components were described by Quintero-Jiménez et al. (2010a). GM liquid medium (Gamborg et al., 1968) amended with 2 % sucrose, 100 mg L^-1 myo-inositol, 1 mg L^-1 pyridoxine, 10 mg L^-1 thiamine, were added to A. tumefaciens strain GV2260 or GV3101 grown to an O.D.₆₀₀= 0 . 8 in a 5:1 (v/v) proportion. Hypocotyls were incubated into this solution for 10 min in soft motion. After this time, liquid was discarded and excess eliminated with sterile paper towels. Explants were placed in co-cultivation medium of induction and multiplication medium (IMM) (Quintero-Jiménez et al., 2010a) added with 200 µM acetosyringone. Explants were incubated in cocultivation medium for 5 d at 25 °C 16 h light (45-70 µmol m^-2 s^-1) and 8 h dark.

Agrobacterium-elimination medium consisted of IMM added with 300 mg L^-1 timentin (GlaxoSmithKline*). Hypocotyls remained into this medium for 10 d under the same growing conditions. Explants were transferred into selection medium wiht the same components as Agrobacterium-elimination medium amended with 50 mg L^-1 kanamycin or 0.2 mg L^-1 glufosinate-ammonium.

Differentiated shoots that remained green in selection medium were separated and grown individually in Magenta* boxes with Elongation and Rooting Medium (ERM) (Quintero-Jiménez et al., 2010a) amended with 50 mg L^-1 kanamycin or 0.2 mg L^-1 glufosinate-ammonium herbicide and 300 mg L^{- 1} timentin to promote elongation and root formation. Growth conditions were identical to the previous stage. In vitro regenerated plantlets were transferred into pots filled with Sunshine* Universal Mix (Sun Gro Horticulture Canada Ltd.) and acclimatized. New plants were placed under greenhouses at 25-28 °C and light intensity of 170-285 (µmol m^-2 s^-1 until maturity.

End point PCR analysis in pdf1.2 and avp1 transfotmed lines

DNA was isolated according to Murray and Thompson (1980) using 100-200 mg of leaf tissue per sample. The final concentration reactions consisted of template DNA (30 ng), primers (0.2 /(M), dNTP's (0.25 mM), Taq DNA Polymerase (1 U), magnesium chloride (2 mM), and Taq buffer (1X). PCR analysis was done using primers to detect 35S promoter pdf1.2, nptII or avp1 genes. Primers for detecting the 35S promoter were sense 35S 5'-GAT AGT GGG ATT GTG CGT CA-3', antisense 35S 5'-GCA CCT ACA AAT GCC ATC A-3', (Invitrogen, Life Technologies). Amplification conditions consisted of 4 min 95 °C for one cycle, 1 min 95 °C, 1 min 54 °C, 1 min 72 °C for 30 cycles and a final extension 7 min 72 °C. The pdf1.2 primers consisted of sense pdf1.2 5'-CAT CAT GGC TAA GTT TGC TTC C-3', antisensepdf1.2 3'-CTC ATA GAG TGA CAG AGA CT-5'; the nptII primers were sense nptII 5'-TCG GCT ATG ACT GGG CAC AAC AGA-3', antisense nptII 3'-AAG AAG GCG ATA GAA GGC GAT GCG-5'. Amplification conditions for both pdf1.2 and nptII primers were 3 min 94 °C for one cycle, 1 min 94 °C, 1 min 55 °C, and 2 min 72 °C for 35 cycles Finally, the avp1 gene primers consisted of sense avp1 5'-ACT GGT TAT GGT CTT GGT GGG T-3'; antisense avp1 5'-GGC AAC ATC TTG CAC AGG GCT GT-3'. Amplification conditions were 5 min a 95 °C for 1 cycle, 1 min 95 °C, 1 min 55 °C, 2 min a 72 °C for 40 cycles and a final extension 7 min 72 °C. The expected fragments sizes were 60 bp for pdf1.2, 700 bp for nptII gene, 195 bp for 35S promoter and 630 bp for avp1. Amplified fragments were separated into 2 % agarose gels and stained with ethidium bromide.

Real time quantitative PCR analysis in T3 pdf1.2 transfotmed lines

Primers and probe were designed using the program Primer Express version 2.0 (Applied Biosystems, Foster City, CA) starting from the sequence of the pdf1.2 gene (NCBI, NM_123809). Defensin gene specific primers and probe (Assays by design, Applied Biosystems) to detect the pdf1.2 gene were sense 5'-AGT TGT GCG AGA AGC CAA GT-3', antisense 3'-GCA TGC ATT ACT GTT TCC GCA AA-5'and the TaqMan* probe 5'-CCC TGA CCA TGT CCC-3' with a 6-FAM™ dye label and minor groove binder (MGB) moiety on the 5' end, and non-fluorescent quencher (NFQ) dye on the 3' end. The internal control 18S ribosomal (4319413E, Applied Biosystem) was tagged with the fluorofore VIC.

Gene exptession analysis

Total cellular RNA was extracted from leaf tissue using Trizol* (Reagent, Carisbald, CA, USA) following the manufacturer instructions. RNA was quantified by fluorometry (TBS-380 Mini-Fluorometer) using the Ribo-Green kit (Molecular Probes, Eugene, Oregon, USA).

First-strand cDNA was synthesized using RT-PCR through One-Step RT-PCR Master Mix (Applied Biosystems; Cat. No.4309169). TaqMan* system was used for gene amplification. The amplification conditions consisted of 48 °C 30 min (Reverse Transcription), 95 °C 10 min (denaturalization), 45 cycles (PCR amplification) at 95 °C, 15 sec and 60 °C, 1 min (ABI PRISM* 7000 Sequence Detection Systems, Applied Biosystems).

The relative expression ratio was calculated by normalizing the target pdf1.2 gene Ct with reference housekeeping gene Ct (18S) to obtain only the efficiency of the pdf1.2 gene (DCt= Ct pdf1.2-Ct18Sr). Each DCt was compared to a calibrator within replicates (DDCt = DCt (sample) - DCt (calibrator). The relative expression was shown as 2^-DDCt, unit of measurement of the expression of the gene (Livak and Schmitrgen, 2001).

Analysis of ptogeny of transgenic plants in T3 pdf1.2 transformed lines

Forty to fifty seeds from selfed common bean transgenic lines were analyzed forpdf1.2 fragment amplification by PCR. Plants that amplified the pdf1.2 or avp1gene fragments were scored and Chi-square analysis was performed to determine segregation ratios and number of insertional events.

Expetimental design

Pinto Saltillo and Flor de Mayo Anita hypocotyls were inoculated with A. tumefaciens strain GV2260 containing pdf1.2 and nptII gene or GV3101 containing avp1 and bar gene in a randomized complete blocks experimental design. Three transformation experiments were carried out and each experiment consisted of three replicates. The experimental unit was one petri dish containing at least 10 hypocotyls. Transformation efficiency was the response trait based on the interaction between cultivars, Agrobacterium strains and selection agents. Data were analyzed by ANOVA and Tukey test (p<0.05) using MINITAB 16 Statistical Software.

RESULTS AND DISCUSSION

Plant transfotmation

The efficiency for transformation of whole plants depends upon the cells being competent for regeneration (Dong and McHughen, 1991; Nagl et al. , 1997), which limited using this technology in common bean. Agrobacterium tumefaciens-mediated transformation of P. vulgaris was developed based on successful regeneration reports previously published (Delgado-Sánchez et al. , 2006; Quintero-Jiménez et al., 2010a, 2010b). The structure derived from germination and enlargement of the embryo develops in hypocotyl structure (Figure 2a). The complex tissue of organogenic shoots formed near the suspensor cells indicates that the full germinative and regenerative potential is translocated to that spot facilitating and promoting the development of two or three shoots, each of them with full capacity to develop a whole plant (Figure 2b). However, not all de novo shoots gave rise to whole plants; in most of the cases only one shoot developed into a full single plant concentrating the morphogenic potential in only one shoot to become a plant (Figure 2c).

Once the organogenic shoots formed, they remained atrached to the hypocotyls until full plant development (Figure 2d). In each fresh medium transfer, a thin layer of the hypocotyl was eliminated until the plant was independent. After full stems and leaves formation and development, roots differentiated at the stem base indicating that the new plant is independent from the hypocotyl, this phase took two-months for full elongation and rooting (Figure 2e).

Plants that maintained a healthy condition were transferred into greenhouse for further observation (Figure 2f). All T0 lines showed similar phenological stages compared to non-transformed plants; they flowered and set seeds at the same rate as control plants (Figures 2g and 2h).

Different strains of A. tumefaciens for inoculation and co-cultivation, as GV2260 and GV3101, induced differential transformation efficiency. With the use of the GV3101 strain some degree of necrosis on the explants and inhibition of full regeneration were observed (Table 1). The A. tumefaciens strain effect was more severe for FMA than for PS reducing the regeneration and transformation efficiency up to 30 % within PS and 80 % in FMA in relation to control plants regeneration (Table 1).

Different transformation trials with A. tumefaciens on FMA and PS showed direct organogenesis response of hypocotyls. Average transformation efficiency (number of resistant shoots to kanamycin / number of explants X 100) for GV2260 (pdf1.2+nptII) was 17 and 28 % for FMA and PS. Transformation efficiency (number of resistant shoots to glufosinate-ammonium herbicide / number of explants X 100) using GV3101 (avp1+bar) was 10 and 21 % for FMA and PS, respectively indicating a statistical difference due to the Agrobacterium strain used (p=0.011) (Table 1).

There was a significant difference (p=0.001) in response for both cultivars to the overall transformation process cutring down up to nearly 50 % in FMA compared to PS regardless the A. tumefaciens strain used (Table 1). The transformation efficiency close to 30 %, suggests that the system herein utilized is comparable, and in some cases superior, to other transformation systems (Aragão et al., 1996, 2002; Kim and Minamikawa, 1996).

The use of kanamycin or glufosinate-ammonium to detect a functional nptII or bar gene in the plant genome was efficient in maintaining the transformed plants in a permanent stage of selection. However, reduced transformation efficiency was reduced (40 %) in both cultivars when using glufosinate-ammonium compared to kanamycin selection (Table 2). Differences in transformation efficiency between cultivars was confirmed using glufosinate-ammonium where FMA showed 3.9 % (p=0.001) compared to PS (8.4 %) (p=0.037) (Table 2).

Genomic analysis

A preliminary analysis of the T0 kanamycin-resistant lines population to verify presence of inserts was performed by end point PCR (Figure 3). These results were confirmed by q-PCR showing amplification of the pdf1.2 gene in the lines that amplified for all the three components mentioned (Figure 4).

The transcriptional expression of the pdf1.2 gene in nearly homozygous T3 population showed that plants expressed different levels ofpdf1.2 gene (0.610.9) which were correlated to levels of tolerance or resistance to Colletotrichum lindemuthianum strains 448 or 1472 (data not shown) (Figure 5).

Segregation analysis

Segregation ratios and χ² analysis indicated that all transgenic lines showed a 3:1 segregation, indicating one construct insertion. The genomic analysis of the plants showed preliminarily an integration of the constructs on T0 plants and evidence of stable integration in T3 populations (Data not shown).

The Agrobacterium-mediated transformation protocol reported here is a viable alternative to incorporate genes into common bean plants for their use in breeding programs. Besides, it is a tool for functional genomics in order to integrate valuable genes or regulatory sequences that may provide added value for agronomical performance, nutritional or market value.

ACKNOWLEDGEMENTS

Authors would like to thank Dr. Miguel Gómez Lim from CINVESTAV-Irapuato, México, and Dr. Roberto Gaxiola Ariza from Arizona State University for providing the gene constructs. This study was financed in part by CONACYT-SAGARPA (109621) and Consejo de Ciencia y Tecnología de Guanajuato (118814).

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