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Published in Crop Sci. 44:1825-1831 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Screening Multiple Soybean Cultivars (MG 00 to MG VIII) for Somatic Embryogenesis Following Agrobacterium-Mediated Transformation of Immature Cotyledons

Tae-Seok Koa, Randall L. Nelsonb and Schuyler S. Korbana,*

a Dep. of Natural Resources and Environmental Sciences, Univ. of Illinois, Urbana, IL 61801
b USDA-ARS, Soybean/Maize Germplasm, Pathology, and Genetics Research Unit, Dep. of Crop Sciences, Univ. of Illinois, Urbana, IL 61801

* Corresponding author (korban{at}uiuc.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A total of 15 soybean [Glycine max (L.) Merr.] cultivars representing maturity groups 00 to VIII were assessed for their embryogenic and transformation potentials. All cultivars were identified as embryogenic under hygromycin selection following Agrobacterium-mediated transformation of immature cotyledons. Histochemical ß-glucuronidase (GUS) assays of induced hygromycin-resistant somatic embryos (SEs) showed that 13 out of 15 cultivars were amenable for transformation. Wide variations among different genotypes were observed for their embryogenic capacity under hygromycin selection. Three cultivars, Cisne, Council, and Kunitz, were highly embryogenic yielding more than 50% responding explants and 1.5 to 2.4 hygromycin-resistant SEs per responding explant. The transformation potential of multiple soybean cultivars was highly correlated with the embryogenic potential of immature cotyledons under hygromycin selection. It was possible to distinguish between highly and poorly embryogenic genotypes by visually observing the phenotype of cultured immature cotyledons. For highly embryogenic cultivars, the induction of somatic embryos mainly originated from actively dedifferentiating and browning/necrotic tissues along the margins of the abaxial side of cultured cotyledons.

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid • GUS, ß-Glucuronidase • HPT, Hygromycin phosphotransferase • SEs, somatic embryos


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SINCE THE FIRST TRANSGENIC SOYBEAN PLANTS were developed via Agrobacterium-mediated transformation of cotyledonary nodes (Hinchee et al., 1988), improvements have been made by means of hypervirulent A. tumefaciens strains, addition of thiol compounds to the cocultivation medium, or sonication of explants (Hood et al., 1993; Torisky et al., 1997; Trick and Finer, 1997; Olhoft et al., 2001). However, successful transformation has been limited to a few soybean cultivars in limited maturity groups (Hinchee et al., 1988; Di et al., 1996; Trick and Finer, 1998; Yan et al., 2000; Donaldson and Simmonds, 2000; Olhoft and Somers, 2001; Olhoft et al., 2003).

An alternative target tissue–transformation protocol that is widely used for soybean transformation is that of proliferative embryogenic suspension cultures–particle bombardment (Finer and McMullen, 1991; Sato et al., 1993; Parrott et al., 1994). These cultures consist of dense globular-stage clumps of embryogenic tissues derived from secondary globular embryos, initially formed on immature cotyledons, and maintained by routine subculture in a liquid medium. However, this regeneration system relies on the lengthy process of liquid propagation before and/or following transformation. Although improvements on the efficiency of the embryogenic culture system and reduced culture time have been reported (Samoylov et al., 1998a, 1998b; Santarém et al., 1998; Santos et al., 1997), this system remains highly genotype-dependent. Moreover, this system is not very desirable because of low transformation efficiency, chimerism, and complex patterns of introduced DNA in transgenic plants (Hadi et al., 1996; Trick and Finer, 1998).

Given that there is no efficient transformation system for a wide range of soybean cultivars, several studies have evaluated the potential of various soybean genotypes for somatic embryogenesis (Parrott et al., 1989; Bailey et al., 1993; Meurer et al., 2001; Tomlin et al., 2002). However, these studies have primarily focused on the embryogenic response of soybean genotypes under nonselective conditions. Meurer et al. (1998) have suggested this strategy may be misleading because infected target tissues may differentiate differently in the presence of Agrobacterium and selective agents. For example, Hartweck et al. (1988) and later Santarém et al. (1997) have reported that when the adaxial side of immature cotyledons is oriented upward on a nonselective medium, high induction of somatic embryogenesis is observed. Whereas, Ko et al. (2003) have reported that frequencies of somatic embryogenesis and transformation are higher when the abaxial side of explants is oriented upward on the selective medium.

In this report, soybean cultivars representing a wide range of maturity groups (MG 00 to MG VIII) have been screened for their embryogenic and transformation potentials using the protocol described by Ko et al. (2003). To our knowledge, this is the first report on screening multiple soybean cultivars of various genetic backgrounds and maturities for somatic embryogenesis under hygromycin selection. We also report on observed phenotypic differences of immature cotyledonary explants that are associated with genotypic variability for embryogenic response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
Fifteen soybean cultivars were selected to represent the range of pedigree diversity in the USA as defined by Gizlice et al. (1996). These cultivars belonged to different maturity groups, based on photoperiod requirements and time to flowering, with lower maturity groups representing more northern-adapted germplasm and higher maturity groups representing more southern-adapted germplasm. The cultivars were Glacier (MG 00), Council (MG 0), MN0301 (MG 0), MN1301 (MG I), Savoy (MG II), Olympus (MG II), NE3399 (MG III), Kunitz (MG III), Cisne (MG IV), KS4895 (IV), Delsoy5500 (MG V), NC Roy (MG VI), Haskell (MG VII), Benning (MG VII), and Cook (MG VIII). Seeds were obtained from the USDA Soybean Germplasm Collection at the University of Illinois. Ten soybean plants from each cultivar were grown under greenhouse conditions with a 15-h photoperiod at 28°C. To induce flowering and pod set for genotypes in maturity groups MG V to MG VIII, a 9-h photoperiod was provided by covering plants with a black screen.

Preparation of Agrobacterium Strain and Plasmid
Agrobacterium tumefaciens strain Chry5 harboring a disarmed derivative of pTiChry5 (KYRT1) was used (Torisky et al., 1997). The binary vector pCAMBIA1305.1 (CAMBIA, Canberra, Australia), containing chimeric genes for hygromycin phosphotransferase (HPT) and GUS with an intron to detect plant-specific GUS expression, was introduced into Agrobacterium strain KYRT1 via electroporation (Duke-Ras and Hookyass, 1995). Bacterial cells were maintained on a solid LB medium supplemented with 50 mg/L rifampicin and 100 mg/L kanamycin.

Cocultivation of Immature Cotyledons and Selection of Hygromycin-Resistant Somatic Embryos
For cocultivation, a single colony from a freshly streaked plate was used to inoculate 5 mL of liquid LB medium containing 100 mg/L kanamycin, and incubated overnight at 28°C. Then, 500 µL of bacterial cells were transferred into 50 mL liquid LB medium, and grown overnight at 28°C on a gyratory shaker at 200 rpm. The bacterial culture was pelleted by centrifugation and then washed with liquid MSD40 medium (Finer and Nagasawa, 1988). Following a second centrifugation, the pellet was resuspended in the same volume of a liquid MSD40 medium containing 100 µM acetosyringone.

The transformation protocol used in this study was a modification of those described by Yan et al. (2000) and Ko et al. (2003). Excised immature cotyledons (5–8 mm) were directly incubated in a bacterial suspension for 1 h without wounding, and then cocultivated with the abaxial side oriented upward on a 0.2% (w/v) GelRite-solidified MSD40 medium (pH 7.0) in the dark at 25°C for 4 d. After cocultivation, explants were transferred to the same medium, but supplemented with 500 mg/L cefotaxime and 10 mg/L hygromycin for a period of 2 wk, and grown thereafter under a 23-h photoperiod provided by cool-white fluorescent tubes (25 µmol m–2 s–1) at 25°C. Explants were then transferred to a fresh MSD40 medium supplemented with 25 mg/L hygromycin, and incubated for an additional 2 wk. Following subculture of explants onto the same selection medium for another 2 wk, explants were evaluated for somatic embryogenesis with a dissecting microscope.

Histochemical GUS Assay
GUS activity was measured histochemically as reported previously (Ko et al., 2003). Hygromycin-resistant SEs, excised from responding explants, were incubated overnight in a GUS assay buffer consisting of 0.1 M NaPO4 (pH 7.0), 0.1 M potassium ferrocyanide, 0.2 M EDTA (pH 7.0), 0.05% (v/v) Triton X-100 with 500 mg/L of 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (X-Glu) (Gold Biotechnology, St. Louis) at 37°C. The number of blue-stained (GUS-positive) SEs was then recorded.

Statistical Design and Analysis
At least 100 immature cotyledons per soybean cultivar were cocultivated with Agrobacteirum strain KYRT1. After cocultivation, 25 immature cotyledons per plate (deemed a replicate) were placed on the selection medium. Approximately 9 to 20 replications per cultivar were used, depending on availability of cotyledons. To evaluate the embryogenic response of explants under hygromycin selection, data were recorded on the number of responding explants developing at least one hygromycin-resistant SE as well as the number of hygromycin-resistant SEs per responding explant. Data were subjected to GLM analysis by the SAS analysis package (version 6.03, SAS Institute Inc.). Means of responding explants were subjected to Duncan's multiple range test. The relationship between the percentage of responding explants and mean number of hygromycin-resistant SEs formed per responding explant was determined by Pearson's correlation coefficient test (r).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Direct somatic embryogenesis under hygromycin selection was mainly initiated on the abaxial side of immature cotyledons within 4 wk after cocultivation with Agrobacterium. Hygromycin-resistant SEs often developed either as single globular, polyembryogenic globular, or as clusters of globular SEs along the margins of cotyledons from all embryogenic genotypes (Fig. 1A, B, and C) . Hygromycin-resistant globular SEs, greenish-yellow to green in color, were readily distinguishable from likely escapes that were yellowish to white in color. However, not all SEs were later deemed transgenic. Two morphologically different types of green globular SEs were observed, including translucent- and opaque-types (Fig. 1A and D). In addition, structures whereby these two types of SEs were fused were also observed (Fig. 1E). Proliferative embryogenic suspension cultures from both types of SEs were established, and these were confirmed as transgenic following histochemical GUS assays (Fig. 1F).



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  		Fig. 1. Somatic embryogenesis on the abaxial side of immature cotyledons under hygromycin selection following A. tumefaciens strain KYRT1-mediated transformation. A, B, and C, somatic embryos formed singly, in clusters, or as polyembryogenic structures, respectively. Histochemical GUS staining of responding explants are shown in (A) and (B). D, an opaque-type globular somatic embryo. E, a fused-type structure of translucent- and opaque-type SEs indicated by arrows. F, proliferative embryogenic tissues derived from an opaque-type SE.

 
By the end of the 6-wk selection period, the genotypic influence on somatic embryogenesis was determined by measuring the percentage of responding explants that formed at least one hygromycin-resistant SE on the selection medium. A hygromycin-resistant SE was characterized as green, globular, and translucent. Unlike translucent-type SEs, individual opaque-type SEs could not be easily separated from explants as they tightly adhered to or were embedded within explants (Fig. 1F). Thus, opaque-type SEs were not scored as hygromycin-resistant.

There were significant differences among all 15 cultivars for the frequency of embryogenic response on the selection medium (F = 60.2, 14 df, p < 0.001). Kunitz had the highest embryogenic response with 54% of inoculated cotyledons developing SEs under hygromycin selection, followed by Council, Cisne, and Savoy with 52, 51, and 24%, respectively (Table 1).


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  		Table 1. Embryogenic response and transformation frequency of explants of 15 different soybean cultivars 6 wk after hygromycin selection.

 
The number of hygromycin-resistant SEs that formed per embryogenic explant was also significantly different among all cultivars tested (F = 15.1, 14 df, p < 0.001). There was a correlation (r = 0.66, p < 0.0001) between the percentage of responding explants and the number of SEs per responding explant. The highest number of hygromycin-resistant globular SEs (average of 2.4 SEs per responding explant) was observed in 54% of immature cotyledons of Kunitz, whereas the number of SEs per responding explant in other cultivars ranged from 1.0 to 2.2 (Table 1). Although Savoy showed high production of SEs on selection medium similar to that of Kunitz, the number of green SEs was lower thus reducing the overall embryogenic response (24%) (Table 1).

To assess the transformation frequency (GUS-positive SEs/tested SEs) for each cultivar, either 50 or 25 SEs from the four highly embryogenic genotypes and all SEs from the remaining cultivars were subjected to the histochemical GUS assay (Table 1). Based on our previous research (Ko et al., 2003), all GUS-positive SEs were confirmed to be transgenic following Southern blotting. Thus in this study, 13 out of 15 cultivars were identified as developing transgenic SEs, and their frequencies ranged from 40 to 76%. All SEs induced from transformed cotyledons of Haskell and NC Roy were identified as GUS-negative (Table 1).

To determine whether differences among cultivars for embryogenic potential corresponded to those for transformation potential, data on percentage of responding explants, induction rate per responding explant, and transformation frequency for each cultivar (Table 1) were subjected to linear regression analysis using estimated values for SE induction and GUS-positive SEs per 100 explants. As shown in Fig. 2 , the frequency of somatic embryo induction (embryogenic potential) among cultivars was highly correlated with the frequency of GUS-positive SEs (transformation potential).



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  		Fig. 2. Relationship between the embryogenic potential and transformation potential of 15 soybean cultivars in the presence of Agrobacterium and selective agents. The embryogenic potential of each cultivar was expressed as the estimated number of hygromycin-resistant SEs per 100 explants (% responding explant x no. of SEs per responding explant). The transformation potential of each of cultivar was expressed as the estimated number of GUS-positive SEs (no. of hygromycin-resistant SEs x % transformation frequency). Values for percent responding explant and % transformation frequency are shown in Table 1.

 
There were no differences among cultivars either for induction period and location of somatic embryogenesis on explants or for morphology of induced SEs on selection medium. However, differences in explant morphology were noted among cultivars, individual plates of the same cultivar, and individual explants within the same plate (Fig. 3 , Table 2). The majority of SEs on selection medium developed along the margins of explants, and these were commonly accompanied with active dedifferentiation and browning–necrosis of tissues. Based on degree of browning–necrosis of tissues along the margins of cotyledons, explants on selection medium were classified into three phenotypes (Fig. 3). These phenotypes included the following: explants with dark-brown necrotic tissues along the margins and collapsed cells in the middle area of the cotyledon (type-A, Fig. 3A), explants with an intermediate phenotype (type-B, Fig. 3B), and explants without necrotic or collapsed tissues (type-C, Fig. 3C). Explants of four cultivars deemed highly embryogenic, including Kunitz, Cisne, Council, and Savoy, mostly displayed type-A phenotypes. Explants of cultivars identified as intermediate in their embryogenic potential (5–10%) usually displayed type-B phenotypes, except for the two genotypes Olympus and Delsoy 5500 that showed mixed type-A and-B and type-B and-C phenotypes, respectively. Interestingly, each of these mixed phenotypes was observed among different plates, but not within the same plate. Explants of cultivars deemed poorly embryogenic (less than 5%) mostly displayed type-C phenotype.



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  		Fig. 3. Representative explants showing three different phenotypes based on degree of cellular response and browning/necrosis along the margins of cotyledonary explants. These were classified as type-A (A), type-B (B), and type-C (C). Nonembryogenic callus formation with or without somatic embryogenesis under hygromycin selection was observed in many genotypes (D).

 

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  		Table 2. Parentage, embryogenic response, and variant phenotypes observed among explants in the presence of Agrobacterium and hygromycin selection of 15 soybean genotypes.

 
Nonembryogenic callus formation along with SE induction was commonly observed in most embryogenic cultivars, but varied for induction period and size (Fig. 3D, Table 2). For Kunitz, inoculated explants under hygromycin selection mainly produced hygromycin-resistant SEs with little callus proliferation during the first 4 wk of induction. Afterwards, nonembryogenic callus formation was often initiated along the edges of explants as the embryogenic response began to diminish (data not shown). For cultivars Cisne and Council, friable, white, and nonembryogenic callus was observed along with SE formation (Fig. 3D). In spite of this undesirable form of callus proliferation, both cultivars remained highly competent for somatic embryogenesis on the selection medium. Whereas, explants of Glacier exhibited rapid swelling followed by vigorous callus proliferation along the cut edges of the explant with low incidence of embryogenesis within 2 wk following cocultivation. Interestingly, Savoy displayed a type-A phenotype and a high number of SEs, similar to other highly embryogenic (>50% responding explants) genotypes, within 4 wk of selection. Then, formation of SEs declined, and was accompanied with overgrowth of nonembryognic callus.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, genotypic effects were observed for induction of SEs on immature cotyledons of soybean incubated on selection medium following transformation. Among 15 cultivars subjected to transformation and selection, the following three cultivars Kunitz, Cisne, and Council exhibited more than 50% responding explants. These embryogenic responses are similar to those reported for highly embryogenic genotypes under nonselective conditions (Parrott et al., 1989; Tomlin et al., 2002). These highly embryogenic responses under selective conditions might be attributed, in part, to the effects of explant orientation (Ko et al., 2003).

Until recently, no efficient screening methods for identifying soybean genotypes with high transformation and regeneration potentials have been available. Owens and Cress (1985) and Delzer et al. (1990) found significant genotypic differences for tumorigenic response of cut stems and wounded cotyledons, respectively, but none of these explants were capable of regeneration. Meurer et al. (1998) reported significant differences among soybean genotypes for their capacity for shoot organogenesis in the presence of Agrobacterium and selective agents. Unfortunately, these differences were not directly related to transformation potential across all genotypes. In this study, a strong correlation between the embryogenic potential and the transformation potential of each genotype, in the presence of Agrobacterium and selective agents, was observed. This indicated that the transformation protocol used in this study was reliable for efficient screening of soybean genotypes for both transformation and regeneration.

Variability among genotypes for somatic embryogenesis has been reported as an inherited trait in many plant species (Chengalrayan et al., 1998; Tar'an and Bowley, 1997). Parrott et al. (1989) have indicated that highly embryogenic soybean cultivars tend to have one or both ancestors that are highly embryogenic. Ko et al. (2003) have also observed similar results with closely related soybean cultivars under selective conditions. ‘Williams’ has been previously reported as highly embryogenic (Ko et al., 2003). In this study, Kunitz, a near-isogenic line of Williams, and Cisne and Savoy, that share the same pedigree that is dominated by contributions from Williams, all have displayed high embryogenic responses with type-A explant phenotypes. Council, which is as highly embryogenic as Kunitz, is derived from ‘Ozzie’ and ‘Dawson’, which are from the same pedigree cluster (Gizlice et al., 1996). MN1301 has a pedigree derived mostly from the two pedigree groups that contain Williams and Council, but is among those cultivars with the lowest percentage of responding explants. The capacity for somatic embryogenesis among soybean genotypes under hygromycin selection seems to be affected by the genetics of the progenitor genotype; however, the specific nature of this genetic control has yet to be determined.

Various studies reported on the effect of maturity group on somatic embryo induction in soybean. Ranch et al. (1985) and Bailey et al. (1993) reported that there was no relationship between maturity and somatic embryogenesis. In contrast, Shoemaker et al. (1991), Tian et al. (1994), and Tomlin et al. (2002) reported that soybean genotypes in early maturity groups (MG 00 to MG I) displayed higher embryogenic responses than those in later maturity groups. However, Shoemaker et al. (1991) attributed this response to the favorable growing conditions available for early maturity groups. In this study, soybean genotypes in maturity groups MG II to MG IV had higher embryogenic responses (F = 30.25, 2 df, p < 0.0001) under hygromycin selection than those in either earlier maturity (MG 00 to MG I) or later maturity (MG V to MG VIII) groups. In general, the maturity group was confounded with parentage. Thus far, it cannot be accurately determined whether the observed effect was due to either maturity or the genetic background of the cultivar. Since Council (MG 0) was among the highest responding genotypes, while KS4895 was among the lowest responding genotypes, these exceptions to maturity trends were present in this genetic material.

The observed differences among cultivars for either embryogenic response or nonembryogenic proliferation were not necessarily due to either genotypic effects or maturity groups, but likely due to the influence of growth conditions of donor plants and/or in vitro culture conditions. Santo and Torné (1986) reported on the influence of the physiological state of the mother plant on tissues grown in vitro. In our previous report (Ko et al., 2003), ‘Macon’ was the least embryogenic cultivar (0.9% embryogenic response) and exhibited a type-C explant phenotype when immature embryos were collected from donor plants grown in the greenhouse during the winter season. In later experiments, explants of immature embryos collected from donor plants of Macon grown during the summer season showed >30% embryogenic response and exhibiting type-A explant phenotype (data not shown). In this study, Council, Kunitz, and Cisne were grown under optimal growth conditions for these maturity groups, and displayed the highest embryogenic response. On the other hand, donor plants of genotypes belonging to late maturity groups (MG V to MG VIII) were grown under a 9-h photoperiod that was provided by covering plants with a black screen. As a result, these plants did not grow as well, and subsequently these genotypes showed lower embryogenic response than genotypes in other maturity groups. However, there were exceptions to these observations. For example, MN1301 (MG I) and KS4895 (MG IV) exhibited low embryogenic responses that were significantly lower than that for Delsoy 5500 (MG V).

The observed morphological differences within some cultivars (NE3399 and Olympus), such as callus proliferation or mixed phenotypes among explants of different plates (Table 2), indicated that in vitro culture conditions influenced somatic embryogenesis of these explants. The presence of microenvironmental differences among different plates; e.g., proximity to light source, humidity, and temperature variations within the growth chamber might all have contributed to variations in embryogenic responses among different plates.

There were large differences in percentage of responding explants, but 13 out of 15 diverse U.S. soybean cultivars were successfully transformed. This indicated that both induction and yield of transgenic SEs under hygromycin selection were most likely genotype-independent. Moreover, observed differences in embryogenic response and somatic embryo production under selective conditions readily corresponded to transformation and embryogenic potentials across most cultivars tested.

Previously, Komatsuda and Ohyama (1988) reported that genotypes with the highest competence for somatic embryo production do not necessarily show the highest rate of embryo conversion to plantlets. In our previous study, about 4% of mature (transgenic) somatic embryos of ‘Jack’ converted into whole plants (Ko et al., 2003). However, adding 5% (v/v) polyethylene glycol or 1.5% (v/v) sorbitol will increase embryo germination and conversion of somatic embryos as reported by Walker and Parrott (2001), and should be used to enhance maturation and conversion of induced SEs of those highly embryogenic genotypes used in this study, including Kunitz, Cisne, Council, and Savoy.


   ACKNOWLEDGMENTS
 
The authors wish to thank Allan Brown for his assistance with statistical analysis, and Betty Leach and Jean Burridge for plant care in the greenhouse. This project was funded by a grant from the Illinois-Missouri Biotechnology Alliance and funding received from the Illinois Council on Food and Agriculture Research-Internal program.

Received for publication October 14, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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