an efficient transformation system in Chrysanthemum[Dendranthema x grandiflorum(Ramat.) Kitamura] fo

An efficient transformation system in Chrysanthemum[Dendranthema x grandiflorum(Ramat.) Kitamura] fo

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Unformatted text preview: 335 Plant Biotechnology, 19 (5), Original Paper 335- 343 (2002) An Efficient Transfornlation System in Chrysanthemum [Dendranthema X grandi lorum (Ramat.) Kitamura Jfor Stable and Noll-chimeric EXpression of Foreign Genes. Harue SHINOYAMAI * , Toshiharu KAZUMA2, Masayasu KOMANO] , Yukio NOMURA3 and Takao TSUCHIYA4 IFukui Agricultural Experiment Station, 52- 21 Ryo-machi. Fukui, Fukui 918- 8215, Japan gFukui Prefectural Horticultural Experiment Station, 35 - 32 - IKugushi, Mihama, Fukui 919 - 1123, .Japan ' Fukui Prefecture Government, 3- 17- I Ote, Fukui, Fukui 910- 8580, Japan 4Fukui Agricultural Extension Office, 3- 16- 10 Matsumoto, Fukui. Fukui 910- 8555, Japan *Corresponding author E-mail address: mik- hal@mta.biglobe.ne,jp Received 28 May 2002; accepted 25 August 2002 Abstract We succeeded in establishing a stable and efficient transformation system of chrysanthemum (cv. consisting Shuho - no - chikara) which could eliminate both the appearance of the chimeric regenerants of transgenic and non - transgenic tissues and that of the transgene inactivated regenerants. compared two transformation systems, callus induction (CI) system and adventitious shoot induction in SI system (SI) system. The transformation frequency in CI systern (4.4%) were higher than that (0.3 %). All regenerated plantlets obtained by CI system express gene stably even after vegetative We gus in propagation. Whilc a few regenerants obtained by SI system have gus gene and express gus gene commercial cultivars of chimeric manner. Then we applied the CI system in other famous and chrysanthemum and obtained transformed plants with high transformation frequency in 15 among 21 cultivars. Regarding the stable gus gene expression in all regenerants, CI system should eliminate gene inactivation in regenerants and is beneficial for the production of genetically engineered chrysan- themum Key words: callus, chimera, chrysanthemum cultivars, gene silencing, gus gene expression to create Introduction Chrysanthemum [Dendranthema x grandiflorum (Ramat.) Kitamural which was introduced into Japan from China during the Nara Era (A.D.710794), become to be one of the important ornamental plants and is propagated vegetatively by stem cutting and in vitro adventitious shoot formation from various tissues and calli (Hill, 1968; Iizuka et al., 1973; Earle and Langhans, 1974; Khalid et al., 1989). Many useful agronomical traits have been intro- into chrysanthemum by conventional cross breeding and selection, and more recently through mutation breeding (Broertjes et al., 1976; Preil et al., 1983; De Jong and Custers, 1986; Dalsou and Short, 1987; Huitema t al., 1987). In conventional cross breeding, hereditary elements from same or other species are combined by sexual reproduction duced completely new gene combinations. How- ever, in cross breeding, utilizable gene resources are limited to related species which are able to be cross pollinated, so genetic diversity is narrow in chrysan- themum. All of the color variants of elite genotypes were obtained by mutation breeding, either sponta- neous or induced, but nlutation breeding has only a limited potential, as modification of existing path- ways. introduction of agronomically interesting by genetic engineering can be an alternative to The traits such breeding methods, especially for vegetative crops. The genetic engineering has a potential to expand the range of genetic variation in chrysan- themum. However, in chrysanthemum, transformation frequency was still low (Renou et al. , 1993; De Jong et al., 1994; Urban et al., 1994), and chimeric plants (Pavingerova et al., 1994; Benetka and Pavingerova, 1995) or the transformants showing inactivation of transgene were widely reported 336 (Pavingerova et al., 1994; Benetka and Pavingerova, 1995; Takatsu et al. 2000). So the aim of this experiment is to establish a stable and efficient Agrobacterium -mediated transformation system of chrysanthemum (D, x grandlflorum) to solve such problems by using the neomycin phosphotransferase gus gene and hygromycin-resistance gene (hpt) were driven by the CaMV 35S promoter and the nopaline synthase (Nos) terminator. Kanamysinresistance gene (nptl was driven by the Nos promoter and the Nos tenninator. The Agrobacterium had been cultured in a liquid LB medium on BIO-SHAKER BR-15 (TAITEC Co. Japan) at 28 ) for 5h. ID gene as a selective marker for G418 resistance and the -D-glucuronidase (gus) gene II (npt as a reporter gene. Transformation using callus induction (CI) system The system reported previously (Shinoyama et al. , 1998) was used after minor modification. Leaf segments were cut from axenic plants by corkborer = 6mm). They were immersed for 30 min Materials and Methods Plant materia Is (c The chrysanthemum [Dendranthema x grandiflorum (Ramat.) Kitamural cultivar 'Shuho-nochikara' was used for establishment of the experimental protocol, and 21 cultivars were used for the application. Shoot tips of plants growing in the greenhouse were surface-sterilized by dipping briefly in 70% ethanol, and then in a 1% sodium hypochlorite solution for 15 min and rinsed 3 times at mg pH 5.8 prior to autoclaving 120 'C for 15 min. The cultures were put at 25 'C under a 16 h photoperiod using cool-white fluorescent lamps or at 25 'C in darkness. The lamps provided a photosynthetic photon flux [PPF (400700nm)]of 60 /1 mol m 2 s 1 EHAIOI (Hood a binary vector plG121 - Hm 1990; Hiei et al., 1994) was supplied by et al. , 1986) harboring (Ohta et al., Dr. K. Nakamura (University of Nagoya) (Fig. 1). EHAIOI has a C58 chromosomal background and a disarmed pTi B0542 (Sciaky et al., 1978). Intron;rZ,a nos P nt H nos-T 35S-P mg green calli medium[MS medium Intron pBIIO1 Construct of T- Hm to plantlet containing 0.5 regeneration mg l-1 - s nos-T 35S P ht nos-T LB DNA of plG121 - Hm. plG121 was constructed from pBIIOI vector (Jefferson et al. 1987), plG221 (35S- P: Intron gus; Ohta et al. 1990) and pLANlOIMHYG (hpt Dr. K. Shimamoto). ; RB. Right border; LB. Left border; nos - P, nopa]in synthase promoter; nos-T, nopalin synthase terminater; 35S-P, cauliflower mosaic virus 35S promoter; npt II, neomycin phosphotransferase gene; Intron, the intron of castor bean catalase gene within the terminal part of the gus gene coding sequence; gus, - D - glucuronidase gene; hpt, hygromycin phosphotransferase gene; prove used for Southern blot analysis of baldigested DNAS (250 - bp PCR product) was indicated bellow the gus gene. N A 6-ben- zylaminopurine (BAP), 0.2 mg l 1 gibberelline A3 (GA3), and 100 mg 1-1 cefotaxime sodium salt Jfor probe(250 bp) 1 were transferred I RB Fig. medium con- Iiquid bcnzylaminopurine (BAP) and 1.0 l-1 casamino g acid] and co-cultivated for 3 days at 25 'C in darkness. The leaf segments were transferred to bacteria elimination CI medium (CI medium containing 250 mg l-1 cefotaxime sodium salt) for elimination of Agrobacterium, and after 10 days, they were transferred to selection CI medium I (CI medium containing 250 mg 1-1 cefotaxime sodium salt and 20 or 30mg 1-1 G418) for selection of putative transformed callus. After 3 subcultures on new selection Cl medium I, the explants were transferred to selection CI medium 11 (CJ medium containing 100 mg 1-] cefotaxime sodium salt and 20 or 30 mg I- I G418) in which the concentration of cefotaxime sodium salt was reduced for promotion of callus proliferation. The leaf segments forming at Agrobacterium infection condition Agrobacterium tumefaciens strain MS induction (CI) medium[MS medium containing 1.0 l- 1 1-naphthylacetic acid (NAA). O.5 1- 1 6- and Skoog, 1962) containing 3% sucrose and 0.3% Gellan Gum (Pure Chemical Co., Japan). The adjusted to 5% Tween in 20 and 50 !lM acetosyringon with Agrobacterium (final OD660 = 0.1). After immersion, the leaf segments were placed onto callus taining in sterile distilled water. The shoot tip explants were cultivated in vitro (meristem culture) on Murashige and Skoog's (1962) basal medium (MS) (Murashige medium was room temperature 337 Table 1) The time l. table for transformation of chrysanthemum. Transformation using the callus induction (CI) system. Procedure Day o CultureAgrobacterium in liquid Immerse into leaf segments MS LB medium Iiquid for medium 5h cantaining Agrobacterium for 30 min Cocultivate leaf segments with Agrobacterium on cocultivation CI mediurrl (End of cocultivation for days) 3 3 medium medium I(selection of putative transformed cells) Transfer to frosh selection CI medium l Transfer to fresh selection CI medium I Transfer to bacteria elimination CI Transfer to selection CI 10 24 38 (Callus induction on the edge of leaf segments) TTansfer to selection CI medium II 52 66 80 Transfer to fresh selection CI G418 Transfer Transfer to fresh plantlet regeneration Transfer to fresh plantlet regeneration 101 122 Collect elongating shoots II (first regeneration medium medium medium (shoot regeneration) collection) and transfer to rooting medium Transfer remaining shoots and calli to fresh plantlet regeneration medium Collect elongating shoots (second collection) and transfer to rooting medium 143 Transfer rooted plantlets to green house 143 - 180 200 onwards Medium medium plantlet - resistant calli to Plants available for testing construction 3%. MS+NAA I Omg 1 1, BA 0.5 mg l ]. Sucrose (Suc. ) Gellan Gum (Gel.) 0.3% MS+NAA 1.0 mg 1 BA O.5 mg l 1. Casamino acids 1.0 g1 Suc. 3%, Gel 0.3% MS+NAA I.O mg l ]. BA 0.5 mg l Suc 3%, Gel. O 3%, Cefotaxime sodium salt (Cef.) CI medium: Cocultivation CI medium: Bacteria elimination CI medium: ll 250 l, l. l, mg BA 0.5 mg 1 1, Suc 3%, Gel. 0.3%, Cef. 250 mg 1 1, G418 20 mg ' 1 Selectlon CI medium I: MS+ IAA 1.0 mg 1 1. l1 l ], Suc. 3%. C.el 0.3%, Cef. 100 ll: 1.0 mg l 1. BA Se]ection CI medium mg l 1. G_.418 20 mg mg l1 l, l. 0.5 mg 1 Plantlet regeneration medium: GAS 0.2 mg 1 Suc. _ %. Gel. 0.4%, Cef. 100 mg Rooting medium: MS+ Suc. 3%, Gel. 0.4%. Cef. ICO mg 1 l MS+NAA MS+BA O5 2) Transformation using adventitious shoot induction (SI) system. Actlvity Day o CultureAgrobacterium in liquid LB medium for 5h 3 segments into MS Iiquid mrdium containingAgrobacterium for 30 min Cocultivate leaf segments with Agrobacterium on cocultivation Sl medium (End of cocultivation for 3 days) 10 Transfer to bacteria elimination SI medium Transfer to frcsh selection SI medium I Immerse leaf Transfer to fresh selection SI 24 38 52 medium I Transfer to fresh selection SI medium Transfer to selection SI medium II Collect elongating shoots (first I collection) and transfer to rocting Transfer remaining shoots to fresh selection SI 66 medium medium II Collect elongating shoots (second collection) and transfer to rooting Medium construction MS+NAA 3%, BA 1.0 mg l ]. Sucrose (Suc. ) Gellan Gum (Gel.) 0.3% MS+NAA 2.0 mg l 1, BA 1.0 mg l 1, Casamino acids 1.0 g 1 1. Suc. 3%. Gel. O.3% MS+NAA 2,0 mg l 1, BA 1.0 mg 1 Suc. 3%. Gel. 0.3%, Cefotaxime sodium salt (Cef.) 2.0 mg l SI medium : Cocultivation SI medium : Bacteria elimination SI medium: l1 250 mg Selection SI Selection SI medium medium Rooting medium: medium Transfer rooted plantlets to green house Plants available for testing 80- 120 140 onwards l, t, MS+NAA 2.0 mg l 1, BA 1.0 mg 1 1, Suc 3%, Gel 0.3%, Cef. 250 mg 1 1. G418 20 mg 1 ll l 1 1. Suc. 3%. Gel 0.3%. Cef. 100 II: MS+NAA 2.0 mg l G418 20 mg mg l 1, BA I O mg I: MS+ Suc.3 l. %, Gel. 0.49lfo. Cef. 100 mg l] 338 obtaining of putativc transformed plantlets. The shoots tips of the regenerated plantlets were cultured on the plantlet regeneration medium for GUS assay and the remaining parts were transferred to rooting medium (MS medium without phytohor- mones) (Table for Southern blot analysis and GUS assay 1). Transformation using adventitious shoot induction (SI) system The leaf segments which were immersed into Agrobacterium suspension as described above, were placed onto adventitious shoot induction (SI) me- dium (MS medium containing 2.0 mg l-] NAA, l.O mgl ] BA and 1.0gl-1 casamino acid) and cocultivated for 3 days at 25 'C in darkness. The leaf segments were transferred to bacteria elimination SI medium (SI medium containing 250 mg l-1 cefotaxime sodium salt) for elimination of Agrobac- 1975) was carried out using a gus gene fragment (250 bp) as a probe (see Fig. 1), with digoxigenin (DIG) Iabeling and CDP-star substrate detection systems (Roche & Boehringer Mannheim, Ger- many) according to the supplier's instruction. G U.S assay' The plantlets which were clonally propagated from a primary shoot by stem cuttings were assayed for expression ofgus gene after incubation with 5bromo- 4- chloro - 3- indolyl - - D - glucuronide (X -gluc.) (Jefferson et al., 1987, Murakami and Ohashi, 1992). The shoots, roots_ (cut into 15 mm in length) and small plantlets (cut into 3 cm) were incubated in 50 mM phosphate buffer (pH 7.2) ImM containing X-gluc., 5mM dithiothreitol (DTT), 0.3% TritonX-100, 5% methanol, 0.5mM potassium ferrocyanide and O.5 potassium ferricyanide for overnight at 37 , After staining, the shoots, roots and small plantlets were rinsed with mM . terium, and after 10 days, they were transferred to (SI medium containing 250 selection SI medium I mg I cefotaxime sodium salt and 20 mg l-1 G418) for selection of putative transformed cells. After 2 subcultures on new selection SI medium I, the explants were transferred to selection SI medium II (SI medium containing 100 mgl-1 cefotaxime sol- and 20 mg l- I G418) in which the concentration of cefotaxime sodium salt was reduced for promotion of adventitious shoot formation. The shoot tips of the regenerated plantlets were cultured dium salt on the plantlet regeneration medium for GUS assay and the remaining parts were transferred to rooting medium (MS medium without phytohormones) for Southern blot analysis and GUS assay (Table 1). Southern blot analysiss DNA Total was extracted frorn 100 mg of fresh leaves of regenerated plantlets or non- transyoung formed control plantlets by the method of Takagi et al. (1993). The leaves were homogenized in liquid nitrogen using a ceramic mortar and a pestle and suspended in Iml of HEPES buffer [0.1 HEPES (pH 8.0), O.1% polyvinylpyrrolidone (PVP) K-30, 4% 2-mercaptoethanol]. After centrifugation at M 10,000g for 5 min at 4 'C, the supernatant was discarded and the pellet was resuspended in new HEPES buffer. This procedure was repeated three times to remove polyphenofs and polysaccharides. Total DNA was isolated from the pellet by sodium dodecyl sulfate (SDS) extraction method described earlier (Honda and Hirai, 1990). The DNA digested with Xbal was subjected to gel -electrophoresis and blotted onto a positively charged Nylon membranes (Roche & Boehringer Mannheim, Germany). Southern analysis (Southern, 70% ethanol binocular. for overnight, then mounted for Results Transforma tion frequenc y The regeneration processes in CI and Sl systems were summarized in Table I and Fig. 2. On CI system, callus induction stage is required at the first step. This caused the longer duration for obtaining of regenerated plantlets as compared to SI system. Regarding the regeneration frequency, plantlets were more easily obtained in SI system than in CI system. Using CI system, 479 Ieaf segments among 3,513 formed G418 resistant calli, with an efficiency of 13.6%, and 153 plantlets were regenerated from the calli on the regeneration medium, corresponding to 4.4% regeneration frequency based on the initial leaf segments. They were obtained after 143 to 180 days of infection with Agrobacterium. On the other hand, using SI system, 979 adventitious shoots were finally obtained from 3,413 Ieaf segments, corresponding to 28.7% regeneration frequency based on the initial leaf segments. They were obtained after 80 to 120 days of infection with Agrobacterium (see Table Then, 2). regenerated plantlets which were obtained by using CI and Sl systems were analyzed by Southern blot analysis to confirm transformation. of all the plantlets was digested The genomic by Xbal, because only one Xbal site is present in the all DNA T- DNA region. In the regenerants obtained by CI system, all the plantlets showed multiple unique bands with the 339 Table 2. Transformation frequency of chrysanthemum cultivar 'Shuho - no - chikara ' in two transformation systems. No. of transformed No. of plantlets* regeneratod plantlet (C) (B) No. of leaf segments cultured Transformation system (A) Callus induction (CI) 3,5 , . *- 123 expressing gus gene ()means No. of of ()means transformation frequencySouthern confirmed by Transformation plantlets in 123(O_ )1 } 4.4(0.0)'") 979 3,413 Adventitious shoot induction(SI) l). 13 Transformation frequency (C/A: %) 45 (3 6) I} 13(O 3)2i . . chimeric manner. plantlets expressing gus gene in chimeric manner blot analysis, was 1 2 3 4 5 6 7 8 9 101112i314 9.4 6.6 Analysis of chimerism All regenerants showing gus gene positive band by Southern blot analysis were assayed by GUS staining for testing of chimerism. Three organs of the regenerated plants, shoots, roots and small 4.4 plantlets, 2.3 2.0 - (k. SI system, blue staining bp) 23.1 - Fig. were used for this experiment. the case of CI system, blue staining was In observed strongly in all the tested shoots and no chimeric shoot was found (Fig. 4A). In the case of 3 Southern blot analy sis of regenerated plantlets. DNA was digested with Xba I and hybridized with a probe of gus gene. Lane 1: Plasmid DNA of plG121 - Hm, used as positive control . Lane 2: DNA from a non- transformant, used as negative control. Lane 3- 6: DNA plantlets expressing gus chimeric manner. The gene plantlets were obtained by adven- from in titious shoot induction (SI) system. Lane 7 - 10: DNA from plantlets expressing gus gene in whole plant. The plantlets were obtained by adventitious shoot induction (SI) system. Lane 11 - 14: DNA from plantlets expressing The gus gene in whole plantlets were obtained by callus plant. induction (CI) system. gus-probe (Fig. 3). On the other hand, in the regenerants obtained by SI system, 45 among 979 plantlets showed multiple unique bands with the gus- probe (Fig. 3), but the remaining 934 plantlets did not show any bands (data not shown). No hybridization signal was detected in non-transformed control (Fig. 3). These results indicate that no escape plantlets by G418 selection were obtained in CI system and all the transformed plantlets had multiple copy of gus gene, but not single copy. was observed in 9 among 45 plantlets having gus gene. The remaining 36 shoots showed mosaic and weak blue staining in a part of the plant, for example, stomata, trichome or cells in the proximal part of petiole (Fig. 4B). Moreover, in the case of CI system, blue staining was observed in all small plantlets and roots, and chimeric or non-staining one was not found (Fig. 4D, G). This blue staining was observed especially in the vascular bundle. Whereas, in the case of Sl system, blue staining without chimerism was observed in 9 among 45 small plantlets and their roots, but the remaining 36 plantlets whose shoots showed mosaic blue staining exhibited again mosaic and weak blue staining or no staining (Fig. 4E. H). Non -transformed control did not show blue staining (Fig. 4C, F, I) All the plantlets obtained by CI system and 45 by SI system were acclimatized, potted in soil and grown in greenhouse. After flowering (six months after acclimatization), the leaves, roots and petals of all the plants were assayed again by GUS staining. In the plants obplantlets obtained tained by CI system, the gus gene expression was observed again in the leaf veins (vascular bundle) and roots, but not in the petals. Moreover, all these plants showed stable gus gene expression in leaf 3 generations of vegetative veins and roots during propagation by stem cuttings. On the other hand, 36 plants originated from plantlets which were obtained by SI system and showed mosaic gus gene expression in the previous assay on the plantlets exhibited again chimeric staining or no blue staining. The remaining 9 plants obtained by Sl system 340 showed blue staining in the entire leaves and roots. In ali the plants obtained by Sl system, no blue staining was observed in petals (Table 2). These full results indicate that efficient method to CI system is a very induce transformants which express stably foreign genes in non-chimeric man- ner even after vegetative propagation. Transformation in various cultivars of chrysan- themum To verify Secondary, we tested the regeneration ability. Six cultivars, 'Yamate-shiro', 'HiroshimaLbeni', 'Kofuku-no-tori', 'Rosanna', 'Utage' and 'Kin-fusha', showed the ability higher than 'Shuho-no- (marked on chikara' 4 and '+++'), cultivars, 'Peach', 'Symbol', 'Bingo' and 'Swan', showed very low ability or no regeneration (marked on ' - '). The remaining 11 cultivars showed same ability to 'Shuho- no-chikara' (marked on '++' or '+' ). Then, we determined the optimum G418 concen- the applicability of CI system for other chrysanthemum cultivars, 21 famous and commercial cultivars were transformed by CI system (see Table 3). At first, we tested the sensitivity to Agrobacterium infection on these cultivars. Five cultivars, tration for selection of transformants. Optimum concentration was 30 mg l- I in 4 cultivars, 'Yamate -shiro', 'Hiroshima-beni', 'Kosuzu', and 'Kin fusha', and 20 mg l-1 in the remaining 17 cultivars. used 20 mgl I G418 for selection of When 'Ohgon--jo', 'Monroe', 'Miss Betty', 'Utage' and 'Kin- fusha' , were more sensitive to Agrobacterium than 'Shuho-no-chikara' (marked on '+++'), and 2 cultivars, 'Peach' and 'Seiun', were less sensitive plantlets to Agrobacterium (marked on '-'). The remaining 14 cultivars showed same sensitivity to 'Shuho- nochikara' (marked on '++' or '+' ). Table 3, Seiun yellow No, of leaf Sensitivity to Regeneration ability 2) segments Agrobacterium infectionl' cuhured (A) 180 80 Yamate - shiro 180 Hiroshima - beni 180 180 180 Kosuzu Kofuku - no - tori Rosanna 180 Snow queen 180 Ohgon - jo Monroe 180 180 180 Miss Betty Utage Kin - fusha 1.80 Pinky 180 180 Peach 180 Sw an Susie Shuho - no - chikara 2) :} which had no gus gene were obtained. used 30 mg l 1 G418 for selection in the 17 cultivars, regeneration of transformed latter plantlets having gus gene was difficult. we transformed these cultivars by CI systerD and the regenerated plantlets were obtained in 15 cultivars. The regenerated plantlets were Finally, CJ418 (mg I l) No, of regenerated transformed Transformation frequency prantlets p]ants (C/'A:9; o) (B) concentration3} (C) No. of + 20 O O ++ ++ ++ ++ ++ + ++ ++ +++ +++ + +++ +++ 20 30 13 13 72 39 39 43 21.7 +++ +++ +++ +++ +++ ++ ++ ++ +++ +++ + + 180 Symbol Bingo Rocky Orange pinky 1) When we 4 cultivars, many escape Transformation frequency of some chrysanthemum cultivars using callus induction (CI) system. Cultivars Surr] mer we transformants in the former 180 180 + ++ ++ 180 180 ++ ++ 180 180 + ++ 30 30 43 5 5 2.8 20 34 34 18_9 20 5 O 5 O 2.8 25 25 13.9 20 25 25 13.7 0.0 20 2 2 20 30 20 20 32 32 17.8 36 36 20 7 o 7 O O O 7 5 O 4 20 2C 20 O O 7 5 20 o 4 20 10 20 + ++ 23.9 20 20 20 + + 0.0 10 11 O 3*9 O.O OO OO 3*9 2. O. 8 O 22 5.6 Judged by No. of GUS blue spots observed per leaf segment after Agrobacterium infection : no spot, +: Ito 10, ++: to 50, +++: 51 to 100 Judged by No of plantlets formed per leaf segment - : no plantlets, +: I to 5, ++: 6 to 10, +++ more than 11. At the concentraticn, Ieaf segments of non - transformed plants could not form any callus. 11 34 1 by GUS assay and Southern blot analysis, and all of them has gus gene and showed full blue staining. The transformation frequency was 1.1 to '_3.9% based on the initial number of leaf segments and 8 cultivars showed higher frequency than 'Shuho-no-chikara'. The highest frequency of transformation was obtained in 'Hiroshima-beni'. In the remaining 6 cultivars which were less sensitive to Agrobacterium and showed low or no regeneration ability, no transformed plants was obtained. tested Discussion Successful transfer of a foreign gene to plants was described in 1985 in tobacco (Nicotiana tabacum) using genetically manipulated strains ofAgrobacterium tumefaciens (Horsch et al., 1985). After that, Agrobacterium has extensively been used to transform a lot of plant species. However, suscepfirst Agrobacterium is different depending on plant species and cultivars and specific knowledge ofAgrobacterium - host compatibility is required for tibility to successful transformation (Godwin et al. 1992) , The susceptibility of chrysanthemum to Agrobacterium tumefaciens (Miller, 1975; De Cleene and De Ley, 1976; Hooykaas et al., 1994) and the genetic variation in the susceptibility among differ- chrysanthemum cultivars on relation to various Agrobacterium strains have been demonstrated (Wordrangen et al., 1991), Although the susceptibility of chrysanthemum to A. tumefaciens is ent widely reported, there are only limited reports indi- cating successful transformation of chrysanthemum in which the introduced foreign gene expressed (Renou De Jong et al., 1994; Urban et al., 1994), and the transformation frequency was still low (about to 12%). Moreover, chimeric I plants consisting of both transgenic and non-transgenic tissues were reported in chrysanthemum (Pavingerova et al. , 1994; Benetka et al., 199_5). We previously reported two different transformation methods to obtain transgenic chrysanthemum by using Agrobacterium, the callus induction (Cl) system and the adventitious shoot induction (SI) system. In this report, we examined whether the two systems for transformation are able to eliminate chimerism and to establish stable expression of foreign genes in the entire plants. As the et al., 1993; Cl system was very excellent to produce transformants which show the stable expresmany sion of foreign genes in the entire plants and to eliminate chimerism, The duration from infection with Agrobacterium to plantlet regeneration in SI system was about 60 days earlier than that in CI system. Then, we tested results, the transformation frequency by Southern blot analysis on all the regenerants. The transformation frequency in CI system was higher than that in SI system. This advantage of CI system overcomes the durational problem. In addition, all the regenerants obtained by CI system have gus gene, but many regenerants obtained by S] system have no gus gene. Next, we analyzed the expression pattern of gus by GUS assay. All the regenerants obtained by gene Cl system expressed gus gene stably in the entire plants. However, almost the plantlets obtained by SI system (934 among 979) did not have gus gene and 36 among the remaining 45 plantlets having gus gene showed gus gene expression in chimeric manner (Fig. 3B). Moreover, we analyzed the expression of gus gene by GUS assay on the plants which were grown in the green house. All the plants obtained by CI system expressed gus gene stably during 3 generations of vegetative propagation by stem cuttings. However, almost the plants obtained by SI system expressed gus gene in chimeric manner (Fig. 3B) or not expressed it. Regarding to the results, it is expected that the plants which are obtained by CI system should express gus gene stably even after sequential generations by vegetative propagation. Inactivation of transgene (silencing) has been observed in chrysanthemum (Pavingerova et al., 1994; Benetka and Pavingerova, 1995; Takatsu et al. 2000). GUS activity level in transgenic chrysan, themurn was 10-fold less than those of tobacco (Daub et al., 1994) and 100-fold less than those of Kalanchoe blossfeldiana (Aida and Shibata, 1996). In addition, Wordrangen et al. (1992) reported that the expression of gus gene driven by C.aMV35S promoter started slowly in chrysanthemum (5 days after infection) as compared to tobacco (2 days after infection). These facts indicate that CaMV35S promoter behaves weak in chrysanthemum than in tobacco. In our experiment, aged plantle,ts which were obtained by CI system showed gus staining only in the vascular bundle (Fig. 3G), but not in petals. This indicates that the use of chrysanthemum necessary for establishing high expression of foreign genes in chrysanthemum. On the other hand, inactivation of transgene was observed in many regenerated plants obtained by SI system (Fig. 3H), but not in those obtained by CI original promoter is system (Fig. 3G). There is a possibility that the showing chimeric gene expression are consisted of the tissues with activated and inactivated transgenes. However, the reason why only the plants obtained by SI system showed chimerism remaines to be clarilified. In either case, CI system plants 34 2 overcomes the problem of chimerism, because all the plants obtained by CI system did not show any chimerism. Moreover, we showed that, using CI system, transformants showing full gus gene expression were successfully obtained in many cultivars of chrysanthemum at the frequency of 1.1 to 23.5%. Thus our CI system is useful and efficient to obtain transgenic chrysanthemum as compared to the previously reported ones. We will create genetically engineered chrysan- themum with some agronomically important genes by using CI system, and modify this systcm to apply to the cultivars which we could not obtain any transformed plant in this experiment. thank Dr. K. Tanaka (Kyoto Prefectural University), Dr. K. Wakasa (National Institute of Crop Science) and Dr. H. Kamada (University of Tsukuba) for valuable discussions and critical reading of the manuscript, and Mr. H. Yamada (Shizuoka Prefectural Agricultural Experiment Station) and Dr. K. Ueno (Kagos.hima Biotechnology Institute) for valuable suggestions. would like to . References Aida. R, Shibata, M , 1996. Transformation of Kalanchoe Agrobacterium tumefaciens and transgenic silencing Plant Sci , 121: 175 - 185 Benetka, V.. Pavingerova. D , 1995. 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An efficient transformation system in Chrysanthemum[Dendranthema x grandiflorum(Ramat.) Kitamura] fo

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