Interactions in the Agrobacterium-soybean system and capability of some Brazilian soybean cultivars to produce somatic embryos

Twenty-five Brazilian soybean cultivars were studied for susceptibility to four strains of Agr bacterium tumefaciens (C58, Ach5, Bo542 and A281) and for their ability to produce somatic embryos. Twelve plants of each cultivar were inoculated in a greenhous e at 46 weeks of age, using 12 inoculation sites per plant. The number of galls formed on plants were counted 8-10 weeks after inocul ation. To study ability to produce somatic embryos, immature cotyledons, 4-6 mm in length, were plated onto N10 medium for induction of somatic embryogenesis, using four Petri dishes with 20 cotyledons for each cultivar. The embryogenic tissues were transferred o nto new N10 medium six times at 15-day intervals and the number of somatic embryos per cultivar determined. Significant interaction bet ween soybean cultivars and A. tumefaciens trains was observed; the most virulent strain was A281. The opine type apparently had no effect on strain virulence, and the most embryogenic cultivars were IAS-5, Cristalina, FT-Cometa, IAC-7 and OC-3. Departamento de Fitotecnia, Faculdade de Ciências Agrárias e Veterinárias, UNESP, Campus de Jaboticabal, 14870-000 Jaboticabal, SP, Brasil. Send correspondence to A.O.M. E-mail: orlando@fcav.unesp.br. Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091, USA.


INTRODUCTION
Soybean (Glycine max L. Merrill) cultivars differ in susceptibility to Agrobacterium tumefaciens strains (Byrne et al., 1987;Delzer et al., 1990;Bailey et al., 1994;Mauro et al., 1995).Although no relationship exists between a given soybean cultivar susceptibility and its somatic embryo susceptibility to A. tumefaciens, information about cultivar susceptibility to A. tumefaciens strains may be useful in studies related to specificity between soybean cultivars and A. tumefaciens strains.
Several studies have shown differences among soybean cultivars in vitro regeneration via somatic embryogenesis (Owens and Cress, 1985;Komatsuda and Okyama, 1988;Delzer et al., 1990;Mauro et al., 1995).Previous studies developed by Bodanese-Zanettini et al. (1993) and Mauro et al. (1994a) found cultivar IAS-5 among the most embryogenic.Successful Agrobacterium-mediated transformation of the soybean depends on embryogenic capability, since somatic embryos can easily be infected and transformed by strains of A. tumefaciens.Few Brazilian soybean cultivars have been studied so far for their interactions with strains of A. tumefaciens or for their ability to produce somatic embryos.The objectives of this research were to study strain and cultivar specificity in the Agrobacterium-soybean system and to identify some Brazilian soybean cultivars with good ability to produce somatic embryos.
The wild-type strains of A. tumefaciens C58 (nopaline), Ach5 (octopine), Bo542 (agropine) and A281 (agropine) were used for inducing gall formation on plants, since according to Petit et al. (1983), different opines could be responsible for pathogenicity observed in tumor formation.The experimental plots consisted of pots containing four plants of each genotype; three replications were used in a randomized block experimental design.The Agrobacterium cultures were initiated on LB solid medium.To prepare the inoculum a small amount of each bacterial colony was taken from the solid medium with a sterile loop and transferred to 250-ml flasks containing 25 ml of LB liquid medium (Sambrook et al., 1989).
To reach log phase growth (OD value ranging from 0.7 to 0.8 at 600 nm) the flasks were incubated overnight at 28 o C in a rotary shaker at 180 rpm.After incubation, the content of each flask was poured into sterile centrifuge tubes and centrifuged at 10,000 rpm (12,062 g) for

Short Communication
Interactions in the Agrobacterium-soybean system and capability of some Brazilian soybean cultivars to produce somatic embryos Antonio Orlando Di Mauro 1 , José Carlos Martins de Nóbrega 1 , Sonia Marli Z. Di Mauro 1 and Glenn Burton Collins 2

Abstract
Twenty-five Brazilian soybean cultivars were studied for susceptibility to four strains of Agrobacterium tumefaciens (C58, Ach5, Bo542 and A281) and for their ability to produce somatic embryos.Twelve plants of each cultivar were inoculated in a greenhouse at 4-6 weeks of age, using 12 inoculation sites per plant.The number of galls formed on plants were counted 8-10 weeks after inoculation.To study ability to produce somatic embryos, immature cotyledons, 4-6 mm in length, were plated onto N10 medium for induction of somatic embryogenesis, using four Petri dishes with 20 cotyledons for each cultivar.The embryogenic tissues were transferred onto new N10 medium six times at 15-day intervals and the number of somatic embryos per cultivar determined.Significant interaction between soybean cultivars and A. tumefaciens strains was observed; the most virulent strain was A281.The opine type apparently had no effect on strain virulence, and the most embryogenic cultivars were IAS-5, Cristalina, FT-Cometa, IAC-7 and OC-3.
10 min at 4 o C. The supernatant was discarded, 15 ml of MSO liquid medium (Murashige and Skoog, 1962) added and the pellet resuspended.

Somatic embryogenesis induction
Plants of each cultivar were cultivated in the field and when immature cotyledons were 4 to 6 mm long 40 pods of each cultivar were collected.Pods were washed thoroughly but gently in a 1% neutral detergent solution, rinsed in deionized water, placed in 70% isopropanol solution for 30 s, and drained.They were then placed in 20% NaOCl for 15 min and rinsed three times with sterile water (Lazzeri et al. 1985).After asepsis, the cotyledons were excised and plated on solid N10 medium (medium MS of Murashige and Skoog, 1962, supplemented with 10 mg/l of NAA) solidified with Phytagel (0.2%).Four Petri dishes (60 x 15 mm), each containing 20 immature cotyledons, were used per cultivar and considered as replications in a randomized block experimental design.
Embryogenic tissue was transferred to fresh N10 medium at 15-day intervals for 90 days, and kept in a growth chamber at 26 o C with a 23/1-hour light/dark photoperiod.Petri dishes containing embryogenic tissue of each cultivar were examined every 15 days for the presence of somatic embryos and the somatic embryo number per cultivar per replication was counted.

Plant inoculations and statistical analysis
Inoculations were performed when plants reached V 8 growth stage (Fehr and Caviness, 1980) using a hypodermal syringe (5 ml).Slight pressure was used to form a drop of bacterial suspension at the tip of the needle, which was used to make a cut across the epidermis and cortex.Four sites per internode, in the three first internodes, approximately 1 cm apart were inoculated, resulting in 12 wound sites per plant.Four to six weeks after inoculation, the plants were scored by counting the number of galls larger than 0.5 cm in diameter, per genotype, per replication, per strain of A. tumefaciens.The suggestions of Mauro et al. (1994b) were always followed, since the authors found better Agrobacterium tumefaciens infection results by performing the inoculations 5 h after inoculum preparation.
The number of galls derived from inoculations as well as the number of somatic embryos were transformed to (x + 0.5) 1/2 and submitted to variance analysis according to procedures suggested by Snedecor and Cochran (1989).Comparisons among means were performed through the Scott-Knott and Tukey tests, at 5% probability level.

RESULTS AND DISCUSSION
The variance analysis results for the number of galls derived from inoculation (Table I) showed differences in soybean cultivar susceptibility and in pathogenicity degree of A. tumefaciens strain in the soybean cultivars.Interaction between soybean cultivars and A. tumefaciens strain was also observed.Similar interactions were previously reported by Byrne et al. (1987), suggesting possible specificity between strains and cultivars.The 17.15% variation indicates reliable results.
The mean number of galls derived from inoculations with each strain of A. tumefaciens on the soybean cultivars (Table II) showed IAS-5 susceptibility to strain A281, FT-Cometa to strain Ach5, and IAC-7 to strain Bo542.IAC PL-1 was susceptible to strains C58 and A281, IAC-4 susceptible to strains Ach5 and Bo542 but resistant to strain C58, and IAC-100 susceptible to strain Ach5 only.Cultivars OC-14, IAC-17, OC-4, Paraná and UFV-14 were resistant to all strains of A. tumefaciens; Peking, as expected, was highly susceptible to all four strains.
Table II also shows that strain A281 was the most virulent, followed by strains Bo542, Ach5 and C58, with no significant differences among them.Bailey et al. (1994) and Mauro et al. (1995) also reported for strain A281 high virulence to the soybean cultivars tested.The results also suggested that additional factors apart from opine type may contribute to pathogenicity degree of the Agrobacterium strains since significant differences were observed between strains carrying the same opine (A281 and Bo542).

ACKNOWLEDGMENTS
Bacterial strains were kindly provided by EMBRAPA-CENARGEN and the University of Kentucky.Research supported by FAPESP and CNPq.Publication supported by FAPESP.

Table I -
Analysis of variance for the number of galls resulting from inoculation of soybean cultivars with four strains of A. tumefaciens (CV% = 17.15;Mean = 1.7127).Significant at the 1% level of probability.d.f.= Degrees of freedom; SS = sum of squares; MS = mean square. *