In Vitro Cell. De. Biol.-Plant 34:8-13. Jan.-March 1998 @ 1998 Societyfor In Vitro Biology 1071-2690/98 $05.00+0.00 SOYBEAN [GLYCINE MAX (L.) MERRILL] EMBRYOGENIC CULTURES: THE ROLE OF SUCROSEAND TOTAL NITROGEN CONTENT ON PROLIFERATION V. M. SAMOYLOV,'D. M. TUCKER,AND W. A. PARROTfl Department of Crop and Soil Scien£es,The University of Georgia, Athens, Georgia 30602- 7272 (Received25 July 1~7; accepted21 October1997; editorG. C. Phillips) SUMMARY To improve proliferation of soyh~an cultures in liquid medium, the effects of sucrose; total inorganic nitrogen; content of NO3-, NH.+, Ca2+, PO.3-, K+; NH.+/NO3- ratio; and medium osmotic pressure were studied using cv. Jack. Sucrose concentration, osmotic pressure, total nitrogen content, and ammonium to nitrate ratio were found to be the major factors controlling proliferation of soybean embryogenic cultures. Growth decreased linearly as sucrose concentration increased from 29.7 mM to 175.3 filM. A sucrose concentration of 29.2 filM, a nitrogen content of 34.9 mM at I to 4 ammonium to nitrate ratio were found to be optimal for the fastest prolif~ration of soybean embryogenic cultures. There was no significant effect on proliferation of cultures when concentrations of NH. +, Ca2+, PO.3-, and K + were tested in the range of 3.50 to 10.50, 1.02 to 3.06, 0.68 to 2.04, and 22.30 to 36.70 filM, respectively. The relative proliferation of embryogenic cultures of four soybean genotypes was evaluated in Finer and Nagasawa medium and in the new medium formulation. Despite genotype-specific differences in growth, the genotypes tested showed a biomass increase in the new formulation equal to 278, 269, 170, and 251 % for Chapman, F138, Jack, and Williams 82, respectively, relative to their growth on standard FN medium. Due to its lowered sucroseand nitrogen content, we are referring to the new medium as FN Lite. Keywor~: embryogenic cultures; carbohydrate content; osmotic pressure; total nitrogen content; ammonium to nitrate ratio; somatic embryogenesis. INTRODUC'110N of other crops. Hence, the availability of a suitable tissue culture technology for soybean embryo proliferation and regeneration may Biotechnologies for plant improv~ment are of interest becausethey be a limiting step for efficient soybeangenetic transformation. can help overcome some of the traditional limitations of crop breed- Therefore, the objective of this study was to identify the roles that ing. Somatic embryo cultures are one of the most important and con- individual medium components have on somatic embryogenesis in venient system~for plant genetic engineering and in vitro propaga- order to improve proliferation of soybean suspension cultures in liq- tion. Therefore, somatic embryogenesis has been reported in many uid medium. Towards that end, the effect of a number of factors, such plant species and extensive studies have been performed to establish as carbohydrate type and concentration, total nitrogen, ammonium, and evaluate factors that may affect the induction and proliferation nitrate, and other macronutrients on proliferation of suspension em- of embryogenic cultures for a number of crops. The importance of bryogenic cultures were evaluated, resulting in the development of carbohydrate source, total nitrogen, ammonium to nitrate ratio, and an optimized medium we refer to as FN Lite. auxin type and lev~l have been identified as major factors affecting MATERIALS AND METHODS the proliferation of embryogenic cultures (for review see Merkle et Initiation and maintenance of cultures. Embryogenic cultures were initi- al., 1995). ated from immature cotyledons as described by Bailey et aI. (1993) except Soybean [Glycine max (L.) Merrill] is the world's most important that the sucrose concentration in the D40 induction medium wa.'!lowered to oil and protein crop, and efforts have been made to develop efficient 3%. Cultures were maintained in liquid FN medium (Finer and Nagasawa, techniques for its in vitro culture and genetic engineering. Such cul- 1988) or in various medium formulations described below, in 125-ml Erlen- meyer flasks on a gyratory shaker (130 rpm) at 260 C under 10 ~Em-2s-1 tures can be a convenient system for genetic transformation of soy- constant li~ht, with a 3-wk subculture period. Cultivar Jack was used as a bean via particle bombardment and recovery of transgenic plants model genotype to test all medium modifications, and results were confirmed (Finer and McMullen, 1991; Sato et al., 1993; Parrott et al., 1994; with Chapman, Jack, Williams 82, and F138, a breeding line derived from Stewart et al., 1996). Although maintenance of soybeanembryogenic Fayette X P14l7l38 (Parrott et aI., ] 994). Conversion of embryos into plants was conducted according to Bailey et aI. (1993). cultures in liquid medium was facilitated by the development of FN Medium refonnulation. The initial reformulation of FN medium began by medium by Finer and Nagasawa (1988) the efficiency of soybean replacing the FN macro salts with modified macro salts of N6 medium (Chu tissue culture manipulations in vitro still remains low relative to that et aI., 1975), modified by addition of CaCI. for a final concentration of 2.0 mM, and 27.9 mM KNO3, 3.5 mM (NH.)2S0., 1.4 mM KH2PO., and 1.5 mM MgSO. (fable 1). The MS micro salts (Murashige and Skoog, 1962), B5 vi- IPresentAddress: Monsanto Company, 700 ChesterfieldParkway N, St. tamins (Gamborg et aI., 1968), L-asparagine, and 2,4-0 (2,4-dichlorophen- Louis, MO 63198. oxyacetic acid) remained as in FN medium. The sucrose content of FN me- 'To whom correspondence should be addressed. dium was lowered to 87.6 mM, which corresponds to 3% (wt/vol). The working 8 NITROGEN AND SUCROSE EFFECTS ON SOYBEAN SOMATIC EMBRYOGENESIS 9 TABLE 1 correspond to 50, 75, 100, 125, and 150% of the nitrate concentration in basal FNN6, respectively. Ammonium content was held constant at 7 mM. MACRO SALTS (roM) COMPOSITION IN MEDIA FOR SOYBEAN Likewise, the amount of (NH.)2S0., was changed accordingly to formulate EMBRYOGENIC CULTURES media with 3.5, 4.75,7.0,8.25, and 9.0 mM of NH.+, which correspond to 50, 75, 100, 125, and 150% of the aromonium content in FNN6 basal me- Media" dium, while holding the nitrate level at 27.9 mM to evaluate the effect of NH. + content on proliferation of cultures. MS FN FNN6 N6 Macro salts In order to evaluate the effect of aromonium to nitrate ratio in FNN6 basal KNO3 18.9 29.9 27.9 27.9 medium, the (NH.)2S0. and KNO3 were changed to 35.0, 28.0, 23.3,17.5, 11.7,7.0, or o mM ofNH.+, and 0,7.0,11.7,17.5,23.3,28.0, or 35 mM of NH.NOg 20.6 9.9 - (NH.),sO. - 3.5 3.5 NO3-, to create media with NH.+/NO3- ratios equal to 1:0, 4:1, 2:1, 1:1, 1:2, 1:4, and 0:1, respectively. However, no attempts were made to compen- CaCl2 2.9 2.9 2.0 1.1 sate the amount of K+ in the media with lower KNO3 content, because such MgSO. 1.5 1.5 1.5 0.8 adjustment would have required the addition of a large aroount of salts not KH2PO. 1.2 1.2 1.4 2.9 normally present in tissue culture media. The effect of K + content on prolif- (NH.):NOg 1:2 1:4 1:4 1:4 Total nitrogen 60.0 49.9 34.9 34.9 eration was evaluated separately. Effect of other macro salts. To evaluate the effect of Ca2+, the amount of oMS = Murashigeand Skoog(1962) medium; FN = Finer and Nagasawa CaCl2 was altered to obtain media with 1.0, 1.5, 2.0, 2.5, and 3.0 mM of Ca2+, which correspond to 50, 75, 100, 125, and 150%, respectively, of the (1988)medium; FNN6 = definedin Materialsand Methods. calcium content in fNN6 basal medium. The effect of PO.3- content was evaluated by altering the amount of KH2PO. in FNN6 basal medium to obtain media with 0.7,1.0,1.7,1.7, and designation for this medium was FNN6. Then, individual components were 2.0 mM of PO.3-, which correspond to 50, 75, 100, 125, and 150% of the modified to determine the optimal concentration of each. The pH was adjusted phosphate level in FNN6 basal medium, respectively. to 5.8 with KOH prior to autoclaving. All media were autoclaved at 1210 C Finally, to evaluate the effect of K+ content on proliferation of cultures, the amount of KNO3 was lowered from 27.9 mM to 20.7 mM, and 3.5 mM and 103 kPa for 20 min. Experimental design. To evaluate proliferation, embryogenic cultures of (NH.)2S0" were replaced with 7.0 mM NH.NO". Therefore, this formulation cultivar Jack first were conditioned in the reformulated media for at least 3 contained the same aroount of total nitrogen, aromonium, and nitrate as FNN6 wk before growth rates were measured. To measure growth rates, 90 to 130 basal medium, although the amount of K+ was lowered from 29.4 to 22.3 mg of embryogenic tissue were placed into each of five flasks containing 35 mM, which corresponds to 76.1% ofK+ in FNN6. Thereafter, 3.3, 7.0, 10.7, ml of the respective media. After 3 wk, tissue from each set of five flasks was and 14.3 mM of KCl were added to obtain media with 25.7, 29.4, 33.0, and combined and its weight was measured. TotaJ biomass increase was calcu- 36.7 mM of K+, which correspond to 87.5,100,112.5, and 125.0% of the Jated according to the following formula: (Wf -Wi)/Wi, where Wi and Wf K+ level in FNN6 basal medium, respectively.