48 1 () Vol. 48 No. 1 2009 1 Journal of Xiamen University (Nat ural Science) Jan. 2009 1, 1 3, 2 (1.,, 361005 ; 2., 730000) :, (DMSO). :DMSO,. 75 mmol/ L DMSO 48 h,, 1. 6 1. 8 ;10mol/ L 48 h,,;,. : ; ; ; :Q 813. 1 :A :043820479 (2009) 0120113206,,. [1 ].,(,, ) [2 ].,,. [1-2 ].,,,.,,,,,,,,,,. :2008204209 :(C0410001), (2007103) 3 :zhuxueyi90 @xmu. edu. cn [3 ], DMSO, /,. 1 1. 1 1. MS 0. 8 mg/ L 2,42D,, (25 1),110120 r/ min [3 ]. 8. 1. 2 Sigma,DMSO ( H PL C ).. 1. 3 Olymp us ( CX31212C02 ), Agi2 lent1100, UV22500,,. 1. 4 1. 4. 1
114 () 2009 50 ml,60 2 g 20 ml, 120 r/ min,12. 3,, 3,. 1. 4. 2 ( Fluroscein diacetate, FDA). 2 mg FDA 1 ml, 50,0. 04 g/ L. FDA 1 1, 10 min,,., = / 100 %. 1. 4. 3,60, [4 ], 80 % ( 1 50), 50 2 h, 3,, 12 000 r/ min 10 min,,,uv22500 250 nm OD. 1. 4. 4,0. 22m,,4,. [ 5 ] : ODS Hypersil C18 ( 5 m, g4. 0 mm 250 mm) ;1. 0 ml/ min. :A 0. 1 %,B 0. 1 % ;B 50 min 15 % 35 % ;250 nm. 1 1 Fig. 1 Comparison of total isoflavone contents in seed, hypocotyls,callus,and suspension cells of J 1 soy2 bean 1. 4. 5 DMSO,,. DMSO,,, 1 2 5 10 mol/ L [6 ]. DMSO,, 75 mmol/ L., 10 50 100mol/ L [7 ]. 24 48 72 h, FDA. 2 () () Fig. 2 Growth curve (left) and total isoflavone contents in suspension cells (right) of J 1 soybean hypocotyl
1 : 115 3 DMSO ( 48 h) A B. ; C D. 5mol/ L ; E F. 75 mmol/ L DMSO A C E ; B D F (40 ) Fig. 3 Light and fluorescent microscopies of FDA stained suspension cells after MJ (10 mol/ L,C and D) and DMSO (75 mmol/ L,E and F) treatment for 48 h and control suspension cells (A and B) A,C,and E : Total cells under white light ; B,D,and F :Living cells appeared green under fluorescent light (40 ) 2 2. 1 1 1 (22 d) (7,) (1) :,1. 55 mg/ g dm ;, 1. 2 2. 1 2. 9,,.
116 () 2009 4 DMSO Fig. 4 Effect of MJ and DMSO on levels of total isofla2 vone in cell2suspension cultures of soybean 5 DMSO Fig. 5 Effect of MJ and DMSO on daidzein contents in cell2suspension cultures of soybean 2. 2,12, 3, 2. :0 3 d ;47 d,6 ;79 d (2 ).,,, (8 ), 4. 88 mg/ g dm (2 ). 2. 3 :,, 6. 2. 3. 1 (6 ), 1 2 5 10mol/ L., 5 mol/ L, 10mol/ L,, 5mol/ L. 24 48 72 h, :(3 A),5 mol/ L 24 h,,, ( p > 0. 05,4) ; 48 h,,,(3c D), (4) ;72 h,,( p < 0. 05, 4),, ( p < 0. 05,5),,. 2. 3. 2 DMSO DMSO,, 75 mmol/ L. :DMSO 48 h, 95 % (3 E F),. DMSO 24 h, ( p < 0. 05), ( p > 0. 05) ;48 h,,7. 54 1. 21 mg, 1. 6 1. 8 (4 5),4. 67, DMSO. 2. 3. 3, 10 50 100 mol/ L,24 48 72 h. 10 mol/ L 48 h,, 1. 3 ( p
1 : 117 6 I. 48 h II. () 10mol/ L 48 h () HPLC Fig. 6 I. Effect of different concentrations of Phe on lev2 els of daidzein in cell2suspension cultures of soy2 bean (treated for 48 h) II. HPL C chromatograms of isolated isoflavone samples f rom suspended cells treated without (upper) or with 10mol/ L Phe for 48 h (lower) < 0. 05,6 I) ;, 38 % (6 ), 1. 43 (), 4. 12. 3, [8-9 ].,,,., [10 ].,10 mol/ L 48 h, 1. 3 ;,,, 138 % (6).,,, [11 ]., [9,12-13 ].,200mol/ L 9 d, 417. 18 % [ 13 ] ;100 mol/ L,10 [9 ].,,. 1. 0 mg/ L,, [14 ] ;110mol/ L,, 20 mol/ L, [6 ]. 5mol/ L, (3C D),,,. DMSO,,, DMSO,,, [15 ]. DMSO, [14,16-17 ]., 75 mol/ L DMSO 48 h, (4 5).,,.
118 () 2009 [3 ], DMSO, 10mol/ L 75mol/ L DMSO, 48 h 4 5,,. : [1 ] Mueller S O,Simon S,Chae K,et al. Phytoestrogens and their human metabolites show distinct agonistic and an2 tagonistic roperties on estrogen receptor a ( ERa ) and ERb in human cells[j ]. Toxicol Sci,2004,80 :14-25. [2 ] Kurzer M S. Hormonal effects of soy isoflavones : studies in premenopausal and postmenopausal women [ J ]. J Nu2 trition,2000,130 :660-661. [3 ],,. [J ]. :,2008,47 (4) :562-566. [4 ],,,. [J ].,2004,23 (1) :45-49. [5 ],. ( HPLC) [J ].,2000,19 (1) :15-20. [6 ],,,. [J ]., 2005,21 (3) :402-406. [7 ],. [J ].,1990,32 (2) :103-111. [ 8 ] Wang Y D, Yuan YJ,Wu J C. Induction studies of methyl jasmonate and salicylic acid on taxane production in sus2 pension cultures of chem Eng J,2004,19 (3) :259-265. Tax us chinensis var mai rei [J ]. Bio2 [9 ],. [J ].,2000,14 (3) :54-56. [10 ],. [J ].,1990,32 (2) :103-111. [11 ],,. [J ].,2004,16 (5) : 473-476. [12 ] Gundlach H,Muller M J, Kutchan T M,et al. Jasmonic acid is a signal transducer in elicitor2induced plant cell cultures[j ]. Proc Natl Acad Sci USA,1992,89 (6) :2389-2393. [13 ],,. [J ].,2006,39 (5) :438-444. [14 ],,. Tween220 Triton X2 100 [J ]., 2004,5 :60-63. [15 ],. [J ]., 1996,48 (1) :34-37. [16 ],,,. [J ]. :, 1997,23 (6) :684-687. [17 ]. [ D ]. :,2002. Effects of Precursor and Elicitor on Isoflavone Accumulation in Cell2suspension Cultures of Soybean L IAN G Xiao2fang 1,ZHU Xue2yi 1 3,L I Hong2fang 2 (1. Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences,Xiamen University,Xiamen 361005,China ; 2. School of Basic Medical Sciences,Lanzhou University,Lanzhou 730000,China) Abstract : The effect s of methyl jasmonate,dimethylsulfoxide (DMSO),and phenylalanine on the growth of suspended cells,accu2 mulation of total isoflavones and daidzein in cell2suspension cultures of soybean were studied. Cell2suspension cultures of soybean were established f rom soybean hypocotyl callus and could accumulated isoflavone stably. The result s showed that dimethylsulfoxide and phenylalanine could promote cell growth and dry matter accumulation. The content s of total isoflavone and daidzein in cell2sus2 pension cultures of soybean were greatly increased when 75 mmol/ L dimethylsulfoxide was added in the culture system for 48 h,be2 ing 1. 6 and 1. 8 times of the control,respectively. Methyl jasmonate,however,could not contribute to the growth of the suspened cells and elicit the accumulation of total isoflavone and daidzein in cell2suspension cultures of soybean. Key words : phytoestrogen ; soybean ;cell suspension culture ;elicitor