Agrobacterium-mediated Transformation Of Chrysanthemum With TFL1 Gene

Chrysanthemum is one of the ten famous flowers originated in China.It is from human cultivation and natural hybridization for many years. Chrysanthemum has greatly vary in respects of leaf, flowertype and disc type,color,fragrance,posture,charm all better.Pass for four flowers man of homour with plum,orchid,bamboo.It is not only cherished and canonize by Chinese,but also far and wide in many countries of world.The kind of chrysanthemum is greatly,there are 3000 varieties of chrysanthemum in China and more than 7000 varieies in the world.Most natural time-flowering of chrysanthemum are concentrately.So the flowering time is the key for the development of chrysanthemum on some apects.develope the variety of flowering time is greatly significant.The TFLI gene is one mumber of the FT/TFL1(FLOWERING LOCUS T/TERMlNAL FLOWER 1)gene family.The gene inhibits the change from the inflorescence meristem to floral meristem,thereby inhibiting the flowering and postponing the flowering-time.The objective of this study is to transfer the TFLI gene to chrysanthemum and to obtain new types of chrysanthemum with characteristic of later flowering through the study of regeneration system and transformation system mediated by Agrobacterium tumefaciens of chrysanthemum,and established a foundation of wide application of improving chrysanthemum cultivars applying transgenic method.The main experiment and results of this paper are such as follows:(1) Study of estable regeneration system and screen transform receptorUsing six Chrysanthemum as explants to obtain in vitro plants.Then through the study of six chrysanthetsum cultivars on regeneration system,the author obtained the efficient regeneration system of 'Guang dong huang' tender leaves,and selected the tender leaves of 'Guang dong huang' as transform acceptor.The regenertion frequency of it is 92 % in medium of MS+2.0 mg/L 6-BA+0.2 mg/L NAA.(2) Construction of the transgenic system of 'Guang dong huang' tender leavesYoung leaves were excised from asepsis chrysanthemum Guang dong huang,cut into 5 mm2,dipped into Agrobaterium(OD600 adjusted to 0.5) for 30 min,co-cultivated 3 days.Then take off bacteria in liquid MS0+500 mg/L Carb+500 mg/L Cef for 1-2 h,delayed to screen 3 days,and screened with 120 mg/L Cef for sterilizing Agrobacterium tumefaciens,15 mg/L Kan for selecting resistant leave.When the resistant shoots were cut down until l cm and then been rooted on 1/2MS+Kan 10 mg/L.(3) PCR and RT-PCR identification of transgenic plantsThe DNA of the first and second generation transgenic plants were anplified by PCR,the result of electrophoresis was that some plants get the same line as the positive control TFLI (998 bp).The PCR results suggested that transgenic has 5 positive plantlets,we principle identify that the target gene has been translated into genome of Chrysanthemum varieties 'Guang dong huang' successfully and the Total conversion is 4.03%.Throug the RT-PCR identificat use RNA of transgenic plants and detected the expression of the exogenous TFL1 gene in the leaves and stem-tips of transgenic plants.(4) Appraising the characteristic of transgenic plants in the fieldBased on observing characteristic in the field,the transgenic plants expressed 7 days later flowering compared with the controlled plants' time-flowering .