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CELL BIOLOGY & MOLECULAR GENETICS

Progeny Analysis of Glyphosate Selected Transgenic Soybeans Derived from Agrobacterium-Mediated Transformation

^a Plant Transformation Core Research Facility, E324, The Beadle Center, Univ. of Nebraska, Lincoln, NE 68588-0665 USA
^b Monsanto Company, 700 Chesterfieldvillage Parkway North, St. Louis, MO 63198 USA

tclement{at}unlnotes.unl.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Glyphosate selection has been implemented into an Agrobacterium-mediated transformation system for soybean [Glycine max (L.) Merr.]. Cotyledonary explants were excised from 5-d-old seedlings and inoculated with an Agrobacterium strain carrying the CP4 gene, which confers tolerance to the herbicide Roundup, and the ß-glucuronidase (GUS) gene. Explants were selected on sublethal levels of glyphosate during shoot initiation and elongation. Glyphosate tolerant shoots were identifiable after 8 to 12 wk of selection. Coexpression of GUS occurred in approximately 80% of the primary transformants (R₀). The R₀ plants were fully fertile, with no apparent phenotypic abnormalities. Progeny analysis from 81 independent transgenic soybean events derived from glyphosate selection demonstrated that the majority of the transformants transmitted the transgenes as a single functional locus.

Abbreviations: AS, acetosyringone • BAP, 6-benzylaminopurine • GA3, gibberellic acid • GUS, ß-glucuronidase • RT, Roundup tolerance

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
BROADENING the gene pool of soybean via plant gene transfer techniques has been accomplished for such traits as herbicide tolerance (Padgette et al., 1995), amino acid modification (Falco et al., 1995), and insect resistance (Parrott et al., 1994). Introduction of foreign traits into crop species requires methods that will allow for routine production of transgenic lines, containing simple inserts. The transgenes should be inherited as a single functional locus in order to simplify breeding. Delivery of foreign genes into cultivated soybean by microprojectile bombardment of zygotic embryo axes (McCabe et al., 1988) or somatic embryogenic cultures (Fine and McMullen, 1991), and Agrobacterium-mediated transformation of cotyledonary explants (Hinchee et al., 1988) or zygotic embryos (Chee et al., 1989) have been reported.

Both Agrobacterium and microprojectile bombardment DNA delivery systems have been successful in generating stable germline events. Microprojectile bombardment has the advantage of allowing for co-transformation of multiple vectors, however, the resultant transformants tend to possess multiple, complex inserts (Birch and Franks, 1991; Tomes et al., 1990). Transformants derived from Agrobacterium-mediated transformations tend to possess simple inserts with low copy number (Birch, 1991).

There are benefits and disadvantages associated with each of the three target tissues investigated for gene transfer into soybean, zygotic embryonic axis (Chee et al., 1989; McCabe et al., 1988), cotyledon (Hinchee et al., 1988) and somatic embryogenic cultures (Fine and McMullen, 1991). The latter have been extensively investigated as a target tissue for direct gene transfer. Embryogenic cultures tend to be quite prolific and can be maintained over a prolonged period. However, sterility and chromosomal aberrations of the primary transformants have been associated with age of the embryogenic suspensions (Singh et al., 1998) and thus continuous initiation of new cultures appears to be necessary for soybean transformation systems utilizing this tissue.

The embryonic axes regenerate via a direct organogenic pathway and thus regenerants are less likely to possess somaclonal variation (Larkin and Scowcroft, 1981). However, protocols using the embryonic axes (Chee et al., 1989; McCabe et al., 1988) do not employ selective agents in the procedures, hence, necessitates scoreable markers or efficient progeny screens to identify transformants. This greatly increases the labor required and ultimately may be cost prohibitive.

Cotyledon explants have the advantage of being amenable to selection and regeneration through direct organogenesis. With cotyledon explants kanamycin has been used successfully in recovering stable germline transformants of soybean (Hinchee et al., 1988), but a high frequency of escape plants were recovered. Our efforts focused on identifying an alternative selective agent for use in the soybean/cotyledon transformation system and to evaluate the inheritance of the transgenes utilizing this system. We report here the use of glyphosate, N-(phosphonomethyl)glycine, the active ingredient in Roundup (Monsanto Chemical Co., St. Louis, MO) as a selective agent within the Agrobacterium-mediated cotyledonary transformation system of soybean and the progeny analysis of 81 independent transformation events derived from glyphosate selection.

	Materials and methods

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Agrobacterium Preparation
The plasmid, pMON21112, contained the uidA reporter gene (Jefferson et al., 1987) under the control of the enhanced 35S promoter (Kay et al., 1987; Odell et al., 1985) and CP4 gene (Barry et al., 1992), which encodes a 3-enolpyruvylshikimate-5-phosphate (EPSP) synthase tolerant to glyphosate. The CP4 gene in pMON21112 was driven by the figwort mosaic virus 35S promoter (Gowda et al., 1989). This vector was used in all transformations. The construct pMON21112 was mobilized into the Agrobacterium strain ABI by triparental mating with the helper plasmid pRK2013 (Ditta et al., 1980). The ABI strain was C58 Agrobacterium carrying the disarmed pTIC58 plasmid pMP90RK (Koncz and Schell, 1986). Transconjugants were selected on Luria Broth plates containing 50 µg mL^-1 kanamycin, 25 µg mL^-1 chloramphenicol, and 100 µg mL^-1 spectinomycin.

Agrobacterium cultures harboring pMON21112 used in the transformations were grown in either YEP medium (10 g/L peptone, 5 g/L yeast extract and 5 g/L NaCl, pH 7.0) or AB minimal salts medium (Chilton et al., 1974). Agrobacterium cultures were pelleted at low speed and resuspended in 1/10 B5/B5 liquid medium (see below) to OD₆₆₀ of 0.3 for use in the inoculations.

Plant Transformation
Seeds of `Asgrow 3237' (A3237) were disinfected with 80% (v/v) ethanol for 2 min. followed by a 15 min wash in 50% (v/v) commercial bleach (NaClO) amended with 2 drops of polysorbate 20. The seeds were rinsed five times with sterile water and immersed in a slurry of Captan [N-(tricholormethyl-thio)-4-cyclohexene-1,2-dicarboximide] for 5 min. The sterilized seeds were placed on hormone-free B5 medium and allowed to germinate for 5 d at 24°C, with a 18/6 h light/dark regime. B5 medium consists of macro and micro nutrients and vitamins described by Gamborg et al. (1968) (Sigma, Cat. #G 5893, St. Louis). All media were solidified with 0.8% (w/v) washed agar (Sigma Cat #A 8678). Explants were prepared for inoculation as previously described (Hinchee et al., 1988).

Explants were inoculated for 30 min. Cocultivation and Agrobacterium resuspension medium consisted of 1/10 B5 medium supplemented with 1.7 mg/L BAP, 0.25 mg/L GA₃, 3% (w/v) sucrose, 20 mM MES(2-[N-morpholino]ethane sulfonic acid) pH 5.4, and 200 µM acetosyringone (AS). Explants were cocultivated for 3 d at 24°C. Following cocultivation, explants were washed in the cocultivation medium containing 500 mg/L ticarcillin and 100 mg/L cefotaxime, without MES and AS.

Explants were placed on shoot induction medium for 4 d prior to glyphosate selection. The shoot induction medium consisted of full-strength B5/B5 medium supplemented with 1.7 mg/L BAP, pH 5.8 and 3% (w/v) sucrose. Cotyledons were placed adaxial side up with the cotyledonary nodal region flush to the medium, amended with sublethal levels of glyphosate ranging from 0.075 to 0.15 mM for 4 wk.

Differentiating explants were subsequently transferred to shoot elongation medium for an additional 4 to 10 wk under decreased glyphosate selection pressure ranging from 0.025 to 0.05 mM. The elongation medium consisted of MS/B5 medium (Sigma Cat #M0404) amended with 1 mg/L zeatin riboside, 0.1 mg/L IAA (indole-3-acetic acid), 0.5 mg/L GA₃, 50 mg/L glutamine, 50 mg/L asparagine, and 3% (w/v) sucrose, pH 5.8. Elongated shoots were rooted, without further selection, on half-strength MS/B5 medium with full-strength vitamins plus 0.5 mg/L NAA (-napthaleneacetic acid) or 0.1 mg/L IAA and 2% (w/v) sucrose.

The antibiotics, ticarcillin and cefotaxime, were maintained within the media throughout selection. Cultures were transferred to fresh medium every 2 wk. Plantlets were acclimated for 1 to 2 wk prior to placement in growth chambers.

Progeny Evaluation
The R₁ progeny derived from selfing 81 independent transgenic R₀ soybean plants were evaluated for GUS expression and tolerance to the herbicide Roundup (RT). Progeny analysis was conducted at the Monsanto research farm at Jerseyville, IL, during the summer of 1994. A single row of R₁ seed from each transgenic plant and control A3237 were sown in each of 3 plots. GUS expression was ascertained, histochemically (Jefferson et al., 1987), approximately 3 wk after planting, by assaying young leaf tissue. Roundup was subsequently applied on two of the three plots at rates of either 2.24 kg/ha (plot 1) or 4.48 kg/ha (Plot 2), while the remaining plot was left unsprayed (Control Plot 3). Plots were visually evaluated for tolerance at 7, 14, and 28 d after spray (DAS) on a scale of 0 to 10. A 10 would be a visual equivalent of the control, unsprayed plot, while a 0 would be equivalent to a dead plant. Segregating RT progeny received a score of 3 or above.

Second generation progeny (R₂) derived from 12 events were evaluated in the greenhouse during the fall of 1994. R₂ seed collected from 6 to 12 individual R₁ plants were sown in flats. Plantlets were numbered and assayed for GUS expression, histochemically, 3 wk after planting. The plants were subsequently sprayed with Roundup at rate of 4.48 kg/ha. Two weeks after the Roundup application, segregation patterns for RT were tabulated.

Molecular Analysis
Southern blot analysis (Southern, 1975) was conducted on five independent primary transformants (R₀) not evaluated in the field study. Total genomic DNA was extracted from three fully expanded leaves following the protocol of Dellaporta et al. (1983). The DNA (10 µg) was digested with KpnI which cuts at a single site within the T-DNA borders of pMON21112 at the 3' end of the CP4 open reading frame which is adjacent to the left border. GUS and CP4 specific probes were generated by random primed synthesis (Prime IT II, Stratagene, cat #300385, La Jolla, CA) incorporating ³²P labeled dCTP. The hybridization was conducted at 60°C in 0.5 M Na₂HPO₄, pH 7.2, 7% (w/v) SDS solution for 14 h. The filter was washed twice in 40 mM Na₂HPO₄, 5% (w/v) SDS, pH 7.2 at 60°C for 15 min each and third wash in 40 mM Na₂HPO₄, pH 7.2, 1% (w/v) SDS at 60°C for 15 min prior to autoradiography. The filter was first hybridized with the CP4 specific probe, stripped by blotting the filter on 3M paper soaked in 0.4 M NaOH and subsequently rinsing twice in 0.1x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) . The stripped filter was then rehybridized with the GUS specific probe under the same conditions outlined above.

	Results and discussion

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Glyphosate Selection
The use of sublethal levels of glyphosate during shoot initiation and elongation provided effective selection within the cotyledonary–node transformation system of soybean. Tolerant soybean shoots were identified after 8 to 12 wk of culture. The tolerant shoots generally required an additional 2 to 4 wk to root prior to transfer to soil. Coexpression of GUS was monitored in 196 (representing approximately 165 independent events) R₀ plants carrying the T-DNA element from pMON21112 (Fig. 1) . A total of 156 plants were co-expressing GUS. The R₀ plants were fertile with no gross phenotypic abnormalities observed. The average seed set per R₀ plant was 262, although there was substantial variation for this parameter (standard deviation ±122).

View larger version (76K): [in this window] [in a new window]   Fig. 1 Coexpression of GUS within a mature leaf of a transgenic R0 plant and a R1 seed (bottom) and a negative control mature leaf and seed (top)

View larger version (82K): [in this window] [in a new window]   Fig. 2 Southern blot of 5 R0 plants and 4 R1 progeny hybridized with a GUS specific probe. Lane 1: HindIII marker; Lanes 2 through 6 represent 5 independent R0 plants (10 µg); Lanes 7 through 10 represent 4 R1 progeny derived from R0-5; Lane 11 represents 10 µg of negative control DNA; Lane 12 represents 10 µg of control DNA spiked with about 36 pg of linearized pMON21112; Lane 13 represents about 36 pg of linearized pMON21112

View larger version (153K): [in this window] [in a new window]   Fig. 3 Segregation of Roundup sensitive R1 progeny under field conditions

The remainder of the segregation patterns observed for GUS and RT did not fit into either of the above three ratios (Table 1 "other" column). A total of 9 events displayed no RT at the levels evaluated (Table 1 "other" column, Table 2) . Of the 17 events that did not fit into one of the three categories for GUS segregation patterns, 14 displayed no GUS expression within their respective R₁ generation and three displayed GUS expression in ratios other than 1:1. Three of the 14 events that displayed no GUS expression within the R₁ generation had RT progeny.

R₂ Progeny Analysis
R₂ seed was collected from glyphosate tolerant plots for six R₀ families that segregated as a single functional locus (Table 4) , four R₀ families that segregated as two independent functional loci (Table 5) and two R₀ families that segregated in a 1:1 ratio (Table 6) . Segregation patterns for GUS expression and RT were ascertained for 12 R₂ individuals across four to six R₁ siblings, and 13-83 R₂ individuals across 12 R₁ siblings for the selected events categorized as harboring one and two independent functional loci, respectively. R₂ segregation data on the selected events that displayed a 1:1 ratio at the R₁ generation was determined on 12 R₂ individuals across 6 to 11 R₁ siblings.

The segregation ratios for the two traits agreed in all but one case R₀ plant number 127, sibling R₁-1 (Table 4). All six events assayed to the R₂ generation had one to three apparent homozygous R₁ siblings. From the sample size evaluated, the R₂ segregation data is suggestive that these lines do carry a single functional locus.

The R₂ segregation pattern for the R₀ plants carrying two independent functional loci, determined on the basis of the sample size analyzed, was expected to identify between three to four R₁ siblings heterozygous at one or both loci and expected to segregate at either 3:1 or 15:1 ratio, respectively. Homozygosity at either one or both loci would be expected to occur in five to six R₁ siblings, and therefore all R₂ individuals would be positive for the scored traits.

Table 5 is a summary of the segregation patterns observed from the seed collected from 12 R₁ siblings, whose respective parental R₀ plants apparently possessed two independent functional loci. The data indicate that none of these events fit a two independent functional loci segregation model.

The R₂ segregation pattern for R₀ Plant Number 121 and R₀ Plant Number 174 are tabulated in Table 6. The R₁ ratios derived from these parental events fit into a 1:1 segregation class. The two possible mechanisms that may have manifested this non-Mendelian segregation category, lack of transmittance of the insert through one of the gametes or chimeric R₀ plant, can be distinguished by analyzing the R₂ progeny. The expected ratio for the lack of transmittance via one of the gametes, would be 1:1 across the seed collected from the R₁ siblings, while a chimeric R₀ plant would result in R₂ individuals segregating in a Mendelian ratio.

The data within Table 6 suggests that the latter of the two mechanisms was responsible for the observed 1:1 segregation pattern at the R₁ generation for Events 121 and 174. However, the data set is inconclusive as to whether these events possess one or two independent loci. On the basis of the sample size of the R₁ siblings, in which seed was collected, there would be two homozygous and four homozygous events captured and four or eight segregating 3:1, for Events 121 and 174, respectively, assuming a single functional locus. The R₂ progeny analysis on Events 121 and 174 did not fit into a 3:1 or a 15:1 segregation class. However, the data is more suggestive of a single functional locus within these two events.

Glyphosate is an effective selective agent for use in soybean transformation. The herbicide is a relatively stringent selective agent at low doses and results in minimal accumulation of phenolic compounds that are typically observed when utilizing the aminoglycoside kanamycin as the selective molecule.

Agrobacterium-mediated transformation generally results in relatively simple inserts within plant genomes as compared with direct DNA delivery protocols (Birch and Franks, 1991). The data in Fig. 2 demonstrates the typical banding pattern that can be expected when using Agrobacterium-mediated transformation protocols.

The progeny analysis conducted indicated that a majority of the lines transmitted the transgene as a single functional locus. In contrast with using the embryonic axis as a target tissue for transformation (Christou and McCabe, 1992), the majority of the R₀ plants recovered were clonal events. The transgenes appeared to be stablely carried on to the R₂ generation and coexpression occurred in approximately 80% of the R₀ events. None of the lines derived from glyphosate selection were sterile and in only one case did the seeds fail to germinate.Finer McMullen 1991

Received for publication January 21, 1999.

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