Genetic Transformation Of A Homeobox Gene PttKN1 In Several Ornamental Plants And Analysis Of Its Functions

Shoot apical meristem(SAM)is the source of plant organ formation and morphologenesis.Among those genes controlled the activity of SAM,KNOXI (KNOTTED1-like homeobox genes)is incontrovertible the most important one.To our knowledge,the functional studies of KNOX gene were focused on the annual herb,while the function of KNOX genes origined from woody plant was still known a little.To get insights into the functions of KNOX origined from woody plant in plant morphologenesis and meristem activity and explore the possibility to improve the properities of the ornamental plant from the transgenic plants,PttKNI(Populus tremula×tremuloides KNOTTED1)gene isolated from the vascular cambium of a hybrid aspen(P.tremula×tremuloides)was introduced into Begonia President-carnot,Beta vulgaris var cicla and Coleus blumei using the leaf disc or floral dipping methods mediated by Agrobacterium tumefaciens in the present study.And the effect of ectopic expressions of the PttKNI on plant morphologenesis was analyzed at the level of morphology,anatomy,physiology and molecular biology.The main results were showed as below:1.PttKNI was tramsformed to B.President-carnot via Agrobacterium mediated leaf disk method,168 primary transformants were obtained screening off kanamycin,which appeared as lobed leaves,cup shaped leaves,abnormal phyllotaxy,ectopic foliole and coral-like plants.Among them,ectopic foliole plants shared enjory value.Results of RT-PCR indicated that the PttKNI was integrated into the genome of B.President-carnot. Observations of paraffin sections and SEM from leaves and stems of transgenic plants suggested that the ectopic meristems formed on the leaf and stem of transgenic plants.2.Floral dip method was used to transform B.vulgaris var cicla.It was the first time of the method used besides the crucifers.There were 132 primary transformants obtained through screening off kanamycin,and 67 plants among them showed purple red and 65 showed green.In the green transgenic plants,phenotypes of plant with three cotyledons, lobed leaves,new stem formed on the inflorescence stem,more inflorescence stem, flattened stem,inflorescence stem with ectopic shoots and green plants with normal morphology shared 0.12%,0.18%,0.18%,0.13%,0.12%and 3.18%,respectively.In red transgenic plants,plant with single cytoledon,three cytoledons,four cytoledons,fused cotyledons,abnormal hypocotyl,accidented leaves,lobed leaves,flattened inflorescence stem,and more inflorescence stem shared 0.18%,0.72%,0.06%,0.12%,0.06%,1.62%, 0.96%,0.26%and 0.06%.Among them,transgenic green plants with normal morphology shared enjory value.Results of PCR indicated that the PttKNI was integrated into genome of B.vulgaris vat cicla;In the green transgenic plants,analysis of endogenous hormone measure and exogenous hormone treatments showed that the endogenous auxin level was corresponded to the wild type,while the endogenous cytokinin level was a little higher; Ultrastructure observation of chloroplast indicated that the number of the granum thylakoid was increased in comparision of wild type;Paraffin sections from transgenic plants with three cytoledons showed that SAM appearance was changed from oblong in wild type to isometry in transgenic plants with three cytoledons,and the xylem vessel of hypocotyl and root in wild type was horizontal,but there were three lines of xylem vessel in transgenic plant with three cytoledon and exhibited 120°between each line;meanwhile, the number of cambium cells reduced in the transgenic plants with flattened stem; Endogenous hormone analysis showed that the hormone level of transgenic plant with three cytoledon was two times of wild type.3.Floral dip method was used to transform C.blumei and 15 plants with kanamycin resistant were obtained.Phenotypes of plants with knots on the abaxial surface of leaves, accidented leaf surface,lobed leaves,clustered shoots and abnormal phyllotaxy(with hexagonal stem)shared 1.0%,0.67%,2.33%,0.67%and 0.33%,respectively.Among them,plant with abnormal phyllotaxy showed tricussate whorled phyllotaxy and enjoyed ornamental vaule.