Establishment Of Rice Transformation System By Regeneration Of Embryogenic Callus Induced From Mature Embryo And MAR (Matrix Attachment Regions) Sequence Mediated Transgene Expression

The continuing development of plant genetic engineering and other related technologies has opened a brand new pathway for crops breeding. As far as the time, many genes that control l~eneficial crop characteristics are being inducted into more and more crop species. Rice, Ori抸a saliva L., one of the most important food crops. is depended Ott by more than 50 percent of people in the world for food. The oriented genetic improvement of rice varieties, which is conducted by plant genetic engineering techniques, stands extremely important significance. It is the coral work of plant transformation to establish high efficient genetic transformation system and to ha~抏 transgenes express at high level. Transgenc silencing and transgene expression variation, however, are two main problems that harass people in transgenic rice research. In this research, with the establishment of high efficient regeneration system by embryogenic callus induced from mature embryo and gene gun mediated transformation, transgenic rice plants that are resistant to basta have been produced. By the transformation ~~ith an expression plasmid that h.3s a MAR-transgene-MAR structure by inserting two MARs at both sides of a bar gene, we tested the influence of MAR sequence mediattion on transgene expression in tr~tnsgenic rice. The result suggests that construction of the MARtransgene-MAR structure can transcriptionally help facilitate bar gene expression, and also enhance the stability of it. As follows are the main results of our research.(1)In this research, using embryogenic callus induced from mature embryo as original material for regeneration, we established a high efficient rice regeneration system that is suitable for genetic transformation by optimizing the combinations of hormones and adjusting the osmotic stresses of the culture media. In our research, we used culture I (LS base,4-D~t)mgll) to induce callus from mature embryo, then cultivated the callus on Culture I1-3(LS base +2,4-D~cmg/I+Sorbitol3g/L), and finally regenerated plants by Cultu rellI-2( MS base 4 NAA0.smgiu+ &BAI.omg/L ?Kinetin2.omgn. ?Sorbitol 8g/L). In the cultivation of embryogtnic callus from 5 varieties of rice by this regeneration system, the regeneration ratio of each variety ranged from 60 to 80 percent. Taking advantage of the high efficient regeneration system that is constructed and optimized in this research to s~t up the rice genetic transformation system, transformed callus gained in the transformation process can regenerate plants high efficiently, and the cffmcicnc~ of rice genetic transformation can be enhanced. Thus to provide the oriented genetic improvement of rice varieties a superb genetic transformation technology systent.(2)Employing cmhryogenic callus induced from mature embryo as acceptors, we set up agcnc gun mediated rice gcnetic transfOrmation system and acquired transgcnic plantsthat arc resistant to b:ISta. TO bc used as acceptors of rice genetic transformation,embryogenic ca1ius has advantages in the convenience of getting original materiaI, thceasincss to induce caIIu:i and thc shortncss of tim f required to get transgenic pI11nts toother materials, which lIave good fcasibiIity in practice.(3) With two special pIant, xpression pIasmids, we studied the effects of MARs on har gcnecxpression in transgcIlic ricc. The result indicated as fOIlows: In the gcnctictransfOrmation of rice vJith MARs mediated bar gcne, the number of transgenic Iincs is66.7 percent higher tha)1 the control, and the number of transgenic plants regcncratedfrom each resistant caIll1s is also increased by 27.3 percent. In the lines tbat bar genc ismediated by MARs, Ie"el of bar gene fxpression is higher than the control, and b1lrgene expression varieb' among transgenic lines is smaIler as well. It is further indicatedb)' this result that thf mediation of A1ARs on transgenes can facilitate transgenecxpression, decreasc or delete transgene siIencing, and thus increase th' amount oftransgcnic lines produced, increase t...