Transgeni根癌农杆菌介导的小麦转基因植株再生_英文_-小麦转基因

1、Received 1998203202, Accepted 1998207201.Project supported by T rans 2Century T raining Programme F oun 2dation for the T alents by Shandong University.T ransgenic Plant R egeneration from Wheat (Triticum aes 2tivum L. Mediated by Agrobacterium tumefaciensXI A G uang 2Min 1, LI Zhong 2Y i 2, HE Chen

2、 2X ia 1, CHE N Hui 2Min 1and Richard Brettell 2(1School o f Life Sciences , Shandong Univer sity ,Jinan 250100,P. R. China ; 2CSIRO Division o f Plant Industry , G. P. O. 1600,Canbberra ,Australia Abstract :A highly efficient trans formation of wheat mediated by Agrobacterium tumef aciens and plant

3、 regeneration is described. The of A. tumf aciens UNN -2fer . and embry ogenic 7genotypes of cultured wheat were used for trans formation. A fter the embry os and calli were co 2cultivated with Agrobacterium and selected with parom omycin (an analogue of kanamycin , resistant embry ogenic calli and

4、resistant plants regenerated from the calli of 3genotypes of wheat were produced. Transgenic plants were determined by PCR am plification of transgene fragments and con firmed by S outhern hybridization with an trans formation efficiency (number of transgenic plants regenerated from independent call

5、i relative to the number of in 2fected calli of 3. 7%5. 9%.The genotypes and the state of a suitable starting material were shown to be im portant in the com petence for T 2DNA trans fer.K eyw ords :Agrobacteriumtume faciens ,wheat(Triticum aestivum L. ,npt gene ,transgenic plantsAgrobacterium tumef

6、aciens has al 2ways been a very useful vector to trans fer foreign genes into plants especially di 2cotyledonous ones because of its highpre 2of 2(H ood 1995 . M onocotyle 2donous plants , particularly the cereals , have been considered outside the host range of A. tumefaciens (Binns 1990 . In recen

7、t years , there has been renewed interest in using the A. tumfaciens sys 2tem to trans form m onocotyledons because of s ome success with rice (Chan et al . 1993,Hiei et al . 1994 , maize (G rims 2ley et al . 1987,Y uji et al. 1996 , onion (Eady et al . 1996 , Asparagus o ffici 2nalis L. (Delbreil e

8、t al . 1993 and bar 2ley (T ingay et al . 1996 etc . , and s ome difficulties encountered in routine trans 2formation method such as protoplast DNA uptake and microprojectile bombardment (Smith and H ood 1995 . There have been several reports about the trans forma 2tion of wheat via Agrobacterium (H

9、ess et al. 1990, Deng et al . 1990,M ooney et al. 1991, Chen et al . 1997 , but from22Acta Phytophysiologica Sinica 1999, 25(1 :22 28only one of which (Chen et al . 1997 transgenic plants were obtained. F or suc 2cess ful trans formation of a plant by A 2grobacterium , it is essential that the ex 2p

10、lant used in cocultivation has the ability to induce Agrobacterium tum or 2inducing (T i plasmid virulence (vir genes. This report describes different genotypes and starting materials of wheat for Agrobac 2terium 2mediated trans formation. The re 2sults suggest that granular embry ogenic calli of wh

11、eat are susceptible to fection.1. 1m aterial Immature embry os were is olated from wheat (Triticum aetivum L. cv. B173, Dra 1, Hesen 3, Bob 3, Xuzhou 211, Jinan 177and 99P 1014days after anthesis and cultured on M B medium(G uo et al . 1991 with 2, 42D 2mg/L (M B2 . Calli formed from these embry os

12、were subcultured in the same medium every 1520days. Before in fection , all emby os and em 2bry ogenic calli were precultured in the M B2medium containing acetosyring one (AS 0. 1mm ol/L for 13days.1. 2B acterial strains and plasmids The Agrobacterium tumef aciens strain used was Agl (Eady et al . 1

13、996 carrying p UNN -2which npt 2to paro 2m (provided by Dr Fiona , CSIRO Division of Plant Industry . Agrobacterium cells were grown on LB medium at 28with appropriate antibiotics (rifam picin 50mg/L and tetracycline 7. 5mg/L for 2days and then washed with M B2liquid medium containing AS 0. 1mm ol/L

14、 and adjusted to op 2timum density.Fig. 1S tructure map of plasmid p UNN -2E:Eco R ; P :P st .1. 3T ransform ation In fection was per 2formed by dipping embry os or embry ogenic calli into bacterial suspension for 30min to 1hour and then trans ferred , without rinsing , to the M B2medium containing

15、AS 0. 1mm ol/L in the dark at 28. A fter co 2cultivation for 2days , the in fected embry o or calli were rinsed in ster 2ile water containing T imentin 150mg/L , blot 2ted dry with sterile filter paper to rem ove bacte 2ria in excess , and then trans ferred onto the se 2lective medium. 1. 4Selection

16、 and regeneration of trans 2form ants The selective medium was M B2containing timentin 150mg/L and paro 2m omycin at 10, 50, 100and 150mg/L. A fter selection for 68weeks , the resistant calli were trans ferred to the M B medium containing indoleacetic acid 0. 5mg/L , zeatin 0. 5mg/L and parom omycin

17、 50100mg/L . Plantlets arising from resistant calli were trans ferred to M B medium containg Pacclobutrozol (PP333 12mg/L and NAA 0. 5mg/L for strengthen 2321期夏光敏等:根癌农杆菌介导的小麦转基因植株再生(英文ing and rooting , and then potted in s oil.1. 5PCR assays and Southern blot analy 2sis T otal genomic DNAs were extr

18、acted from y oung leaves of resistance and control plants according to the CT AB (cetyltriethylamm onium bromide method.PCR (polymerase chain reaction assays were carried out to detect the npt gene in the resistant plants. The PCR reaction was per 2formed in a v olume of 20l containing 30ng sam ple

19、DNA or plasmid (for positive control , 50pm ol of each olig onucleolide primer , 250m ol of dNTP , 1U of promega and PCR bu ol Tris 2, 0K Cl ol a of 95for and 35repeats of 95for 20s fol 2lowed by 60for 2min and 74for 2min.DNAs of s ome plants were analyzed in m ore detail using S outhern hybridizati

20、on method.F or S outhern blots , 20g of is olated total genomic DNA of transgenic wheat plants and untrans formed control plants was digested with Eco R restriction enzymes and separated by electrophoresis in agarose gels. The gels were blotted onto Magna G raph (Micron Separation Inc. nylon membran

21、es. The membranes were hybridized with npt probe which was labeled as EC L T M direct nucleic acid labeling and de 2tection systems (Amersham Life . A f 2ter filter ray for R esults2. 1T ransformation of wheat withA. tumefaciensT able 1shows the data from 6inde 2pendent experiments in which 7geno 2t

22、ypes of wheat were used for A. tumefa 2ciens 2mediated trans formation.T able 1Different genotypes of embry os and calli used for Agroin fection and their selective efficiency Exp. G enotype Embry o (E Age of Number of Number of E fficiency of (1B206E 3d 16001B173E 3d 7302Dra 1E 1d 11002B206E 1d 620

23、3B206C 7d 9703B206C 10d 4904B206C 15d 5204B206C 25d 7104Xuzhou 211C 1m onth 3804B173C 2m onth 5846. 9599P C 1a 9899. 15Jinan 177C 2a 80 56. 26Jinan 177C2a4219021. 4Immature embry os of B173, B206and Dra 1cultured for 1and 3days and embry ogenic calli (Fig. 22 derived from immature embry os of B206,

24、B173,42植物生理学报25卷Xuzhou 211, Hesen 3, 99P and Jinan 177genotypes ranging in age from 7days to over 2years were tested for agroinfec 2tion (T able 1 .Fig. 2T rans formation of embry ogenic calli and m olecular analysis:The starting T ype embry ogenic calli of Jinan 177used for agroin fection. :Resista

25、ntcalli of Jinan 177formedon M B2medium containing parom omycin 100mg/L after selected for 30d. :Resistantcalli of 99P starting to dif 2ferentiate after 7d of culture on the differentiated medium with parom omycin 50mg/L. :Transgenic plantlets of Ji 2nan 177N o. 84after 30d of culture on the differe

26、ntiation medium with parom omycin 50mg/L. :PCRanalysis of re 2sistant plantlets of Jinan 177and control. The bands correspond to 680bp npt gene sequence. 17:T ransgenic plants ; +:P UNN -2; -:Untrans formed plant ; M :M olecular weight marker (S pp1DNA/Eco R fragments . :S outhern blot hybridization

27、 analysis of trans formed wheat plants and controls. All plants were digested with Eco R . 1,3,5: p UNN -2(1 , 99P N o. 14(3 and Jinan 177N o. 59(5 ; 2,4:Untrans formed wheat plants 99P (2 and Ji 2nan 177(4 ; M :M olecular weight marker ; :3.0kb fragment .521期夏光敏等:根癌农杆菌介导的小麦转基因植株再生(英文 A fter being c

28、o 2cultivated withp UNN -2/Agl for 23days at 28, embry os and embry ogenic calli were placed on the selection medium as de 2scribed in Material and Method. S ome of the resistant calli with yellow , granulated structure were produced from the inocu 2lated calli of Hesen 3, 99P , and Jinan177(Fig. 22

29、, but none was formed from embry os (T able 1 after 24weeks of selective culture during which they became white and s oft The resistant 22from 6. 2%to 4%(number of resistant calli /number of calli used for Agrobacteria in 2fection (T able1 .A fter selection , the parom omycin 2resistant calli from a

30、ll varieties were trans ferred to the differentiation medium containing parom omycin 50100mg/L where shoot initiation and subsequently plantlet formation (Fig. 22, took place. The frequency of regeneration was 67%for 99P and 100%for Hesen 3and Jinan 177. M ore than one hundredparom omycin 2resistant

31、 plants were pro 2duced. These plants were strengthened as described in Material and Method , and then trans ferred to s oil , s ome of which have survived and overwintered.2. 2PCR analysis of resistant plantsM olecular analysis of resistant plants used as selective markers by PCR am plification of

32、DNA from sam of pro 22of from all plants resis 2tant to parom omycin , the PCR am plifica 2tion was repeated 23times.Fig. 22shows the am plified 680bp characteristic fragments of npt gene from s ome of the resistant plants. The re 2sults revealed that long 2time 2subcultured embry ogenic calli of wh

33、eat susceptible to Agrobacterium infection. The trans forma 2tion efficiency was between 3. 7%and 5. 9%(T able 2 which were similar to that in s ome of the m onocotyledons and m ost of dicotyledon plants mediated byAgrobacterium.T able 2The efficiency of wheat trans formation mediated by A. tume fac

34、iens Agl based on PCR analysisG enotype Number of resistantplants/number of calli differentiatingNumber of plants analyzedNumber of positive plants /number of indepemdent calliE fficiency 3(%Jinan 17712/5125/33. 799P 20/62012/54. 0Hesen 33/333/35. 1Jinan 177105/848424/845. 93:E fficiency of trans fo

35、rmation was calculated as the number of positive plants regenerated from independent resistant calli/calli used for in fection (see T able 1 .62植物生理学报25卷1 期 夏光敏等 : 根癌农杆菌介导的小麦转基因植株再生 ( 英文 27 Successful transformation of wheat cultures is dependent on many facteors , of which the selection of suitable

36、 geno2 type and starting material are of particu2 lar importance. The immature2embryos2 derived Type embryogenic calli ( Guo et al . 1991 subcultured for half to two years which were compact clusters of or2 ganized cells and golden yellow in color were shown to be excellent starting mate2 rials in a

37、ll of our transformation experi2 ments. Chen et al . ( 1997 recently re2 ported Agrobacterium2mediated transfor2 mation of wheat using freshly isolated im2 mature embryos , precultured immature embryos and embryogenic calli of Bob2 white , and no significant difference in the transformation efficien

38、cy was shown in the three explant types , but several experiments with all three explants actu2 Many resistant plants have not been transformed , which included even differ2 ent plants coming from the same resistant callus. Therefore the resistant calli must be divided into small enough ones in sub2

39、 culture for selection so as to reduce the chance that untransferred cells go unno2 ticed. 2. 3 Southern blots analysis The plasmid vector pUNN - 2 was re2 stricted with EcoR , to generate 3. 0 kb fragments containing the npt gene. Hybridization with the npt gene probe detected on 3. 0 kb fragments

40、in EcoR 2 restricted transgenenic plant genome DNA and p UNN - 2 , indicated that the npt gene had been transferred to wheat plants ( Fig. 22 . 3 Discussion ally failed to produce any transgenic plants , the reason for which was not clear. We think the physiological state of the starting material of

41、 wheat can affect the success of agroinfection. The choice of starting concentration of paromomycin for the transformation of wheat calli is also important. The fre2 quency of resistant calli produced was 6. 2 % 21. 4 % when paromomycin 10 mg/ L was used for the first and (or sec2 ond selection , bu

42、t only 1. 5 % 2 % on a starting selection medium containing paromomycin 50100 mg/ L ( not shown in Table1 . The higher transformation frequencies ( 3. 7 % 5. 9 % obtained here than the other report ( Chen et al . 1997 might also be owing to the use of stronger constitutive promoter Ubi 1 as was repo

43、rted for transformationin of other monocotyledons ( Li et al . 1997 , Tingay et al . 1996 . The length of co2cultivation period affected the efficiency of transformation. A co2cultiture time of 2 3 days was needed so that there were enough the bacteria to cover the calli to be infected , so that hig

44、hly resistant calli and trans2 formed plants can be obtained. Similar results were also reported by Chen et al . In their experiments , where G expres2 US sion was detected after 2 or 3 d of co2cul2 ture ( Chen et al . 1997 . In summary , by using long culture time on embryogenic Type calli and Agro

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