Agbiogeneric soybean with glyphosate tolerance: Genetic transformation of new Colombian varieties

Soybean (Glycine max L.) is a major global crop. Genetic modification of this crop can contribute to enhance yields, reduce pesticide use, and facilitate sustainability, but there is a need for locally adapted genetically modified varieties. Glyphosate tolerant soybean developed under an agbiogeneric model is an attractive prospect as the technology is now mostly in the public domain. This research evaluates the potential for transformation of three soybean varieties (BR1, BR2, and FNS) and an established variety (SK7). The study investigates the interaction between these genotypes and two A. tumefaciens strains, establishes a selection protocol using glyphosate, compares two explant types and evaluates a bacterial removal protocol. Our findings highlight the importance of plant genotype and bacterial strain specificity in the transformation process, with variety BR1 consistently showing the best performance. While challenges remain, this study advances in the development of glyphosate-tolerant soybean varieties as agbiogenerics.

Keywords:
Herbicide tolerance; Public domain; Agrobacterium tumefaciens; Glycine max; GM crops

Authorship

Julian Mora-Oberlaender ^** E-mail: jomorao@unal.edu.co

Universidad Nacional de Colombia, Grupo de Ingeniería Genética de Plantas, Carrera 30 # 45-03, Bogota, Colombia. Universidad Nacional de ColombiaColombiaBogota, Colombia Universidad Nacional de Colombia, Grupo de Ingeniería Genética de Plantas, Carrera 30 # 45-03, Bogota, Colombia.

https://orcid.org/0000-0003-0304-2380

Yadira Rodriguez-Abril

Federación Nacional de Cultivadores de Cereales, Leguminosas y Soya, kilómetro 1, vía Cota Siberia, Vereda El Abra Cota, Cundinamarca, ColombiaFederación Nacional de Cultivadores de Cereales, Leguminosas y SoyaColombiaCundinamarca, Colombia Federación Nacional de Cultivadores de Cereales, Leguminosas y Soya, kilómetro 1, vía Cota Siberia, Vereda El Abra Cota, Cundinamarca, Colombia

Meike Estrada-Arteaga

Luisa Galindo-Sotomonte

Instituto de Genética, INTA - Instituto Nacional de Tecnología Agropecuaria, El Ñandú y Gabriel de Aristizábal s/n, Provincia de Buenos Aires, ArgentinaINTA - Instituto Nacional de Tecnología AgropecuariaArgentinaProvincia de Buenos Aires, Argentina Instituto de Genética, INTA - Instituto Nacional de Tecnología Agropecuaria, El Ñandú y Gabriel de Aristizábal s/n, Provincia de Buenos Aires, Argentina

Juan David Romero-Betancourt

Jenny Paola Jiménez-Barreto

Camilo López-Carrascal

Universidad Nacional de Colombia, Facultad de Ciencias, Departamento de Biología, Carrera 30 # 45-03, Bogota, ColombiaUniversidad Nacional de ColombiaColombiaBogota, Colombia Universidad Nacional de Colombia, Facultad de Ciencias, Departamento de Biología, Carrera 30 # 45-03, Bogota, Colombia

Alejandro Chaparro-Giraldo

* E-mail: jomorao@unal.edu.co

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (4)
Tables (1)

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Figure 1
GUS expression in A. tumefaciens infected soybean explants. A, GUS positive explants for each soybean variety and bacterial strain used. Different letters denote significant differences between values. B, Soybean BR1 explants assayed for GUS expression after cocultivation with A. tumefaciens strain AGL0. Top: Explants where expression was detected after cocultivation with A. tumefaciens AGL0 at OD₆₅₀ 0.6, 0.8 and 1.0; NC: Negative control, explant not cocultivated; NT: Cocultivated explant where no GUS expression was detected.

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Figure 2
Elongation efficiency of explants from three soybean varieties under four different glyphosate selection schemes. Control: no glyphosate; T1: 50/12; T2: 100/24; T3: 150/36 and T4: 300/72 μM. Different letters denote significant differences between values.

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Figure 3
Regeneration efficiency for soybean explants from four varieties at four stages (A, Shoot induction; B, Shoot elongation; C, Plantlet rooting; and D, Plantlet hardening). C0: Explants not subject to cocultivation or selection; CG: explants not subject to cocultivation but under selection scheme 2; CC: explants subject to cocultivation and selection scheme 2. Different letters denote significant differences between values.

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Figure 4
PCR analysis of soybean DNA (see Table 1). A. Soybean endogenous lectin gene, 50bp ladder. B. cp4epsps transgene, 50bp ladder, PC: positive control (pEIGP plasmid). C. A. tumefaciens pTiBo542 plasmid, 100bp ladder, PC: positive control (AGL0 DNA). Lanes 3-8 potentially transformed plants. CP: non-transformed plant. NC: negative control.

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Table 1
Primer sequences and PCR conditions

Figure 1 GUS expression in A. tumefaciens infected soybean explants. A, GUS positive explants for each soybean variety and bacterial strain used. Different letters denote significant differences between values. B, Soybean BR1 explants assayed for GUS expression after cocultivation with A. tumefaciens strain AGL0. Top: Explants where expression was detected after cocultivation with A. tumefaciens AGL0 at OD₆₅₀ 0.6, 0.8 and 1.0; NC: Negative control, explant not cocultivated; NT: Cocultivated explant where no GUS expression was detected.

Figure 2 Elongation efficiency of explants from three soybean varieties under four different glyphosate selection schemes. Control: no glyphosate; T1: 50/12; T2: 100/24; T3: 150/36 and T4: 300/72 μM. Different letters denote significant differences between values.

Figure 3 Regeneration efficiency for soybean explants from four varieties at four stages (A, Shoot induction; B, Shoot elongation; C, Plantlet rooting; and D, Plantlet hardening). C0: Explants not subject to cocultivation or selection; CG: explants not subject to cocultivation but under selection scheme 2; CC: explants subject to cocultivation and selection scheme 2. Different letters denote significant differences between values.

Figure 4 PCR analysis of soybean DNA (see Table 1). A. Soybean endogenous lectin gene, 50bp ladder. B. cp4epsps transgene, 50bp ladder, PC: positive control (pEIGP plasmid). C. A. tumefaciens pTiBo542 plasmid, 100bp ladder, PC: positive control (AGL0 DNA). Lanes 3-8 potentially transformed plants. CP: non-transformed plant. NC: negative control.

Table 1 Primer sequences and PCR conditions

Target	Primers	Sequence	Amplicon length (bp)	Amplification conditions
Soybean Lectin	Le1-F/R (Schmidt and Parrot 2001Schmidt MA, Parrot WA2001 Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction. Plant Cell Reports 20:422-428)	5′ TCCCGAGTGGGTGAGGATAG-3′	66	94 °C x 2 min, 35 x (94 °C x 30 s, 54 °C x 30 s, 72 °C x 30 s), 72 °C x 5 min
		5′ TCATGCGATTCCCCAGGTAT-3′
cp4epsps (mod.)	2J-F/R (Rojas et al. 2018Rojas A, López-Pazos S, Chaparro-Giraldo A2018 Screening of Colombian soybean genotypes for Agrobacterium mediated genetic transformation conferring tolerance to glyphosate. Agronomía Colombiana 36:24-34)	5´CTTTGCTGAAGGAGCTACCG-3′	205	94 °C x 2 min, 35 x (94 °C x 15 s, 54 °C x 45 s, 72 °C x 30 s), 72 °C x 10 min
		5´GTGATCGAGATGCGTAGCAA-3′
pTiBo542	pTiBo542-F/R (Deeba et al. 2014Deeba F, Hyder MZ, Shah SH, Naqvi SM2014 Multiplex PCR assay for identification of commonly used disarmed Agrobacterium tumefaciens strains. SpringerPlus 15:358-364)	F: 5′-CCCGCTGAGAATGACGCCAA-3′	766	94 °C x 3 min, 35 x (94 °C x 20 s, 61 °C x 20 s, 72 °C x 40 s), 72 °C x 20 min
		R: 5′-CCTGCGACACATCGTTGCTGA-3′

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[1] * E-mail: jomorao@unal.edu.co