Salt-tolerance Transformation Of Liquidambar Formosana L. Mediated By Agrobacterium With A Positive Selection System

Soil salinity is a serious problem worldwide. Tree can not only improve entironment butalso bring with economic benefits. So breeding varieties with high salt tolerance is urgentaffairs to us. Genetic engineering breaks the limitations of conventional breeding methods,which can transfer salt-tolerance gene directly into tree organism and improve forest breedingprogress. But the safety of selectabe marker gene has caused a widespread pulic concern. Inthis study, a positive selection system was used for salt-tolerance transformation of Chinesessweetgum(Liquidambar formosana L.) and three results were obtained as follow:(1) A double T-DNA vector pCAMBI1301-NHX was constructed.We are successful to construct the plant expression vector pCAMBI1301-NHX thatcontains phosphomannose isomerase structural gene (manA) as selectable marker andsalt-tolerance gene AtNHX1.These two genes were cloned into the two separate T-DNA regionof the vector. Through genetic transformation mediated by Agrobacterium, transgenic plantswere obtained by mannose selection. ManA gene and AtNHX1 gene will be separated in T1lines, marker-free transgenic plants will be obtained.(2) It is the fist time that we established a mannose seletion system for Chinese sweetgumtransformation.The result of explant sensitivity to mannose showed that regeneration rates and number ofshoots per explant decreased with increasing mannose and decreasing sucrose concentration. Itmeans that leaves of Chinese sweetgum can not use mannose as carbon source to induce shootsand we can use mannose as selective agent for Chinese sweetgum transformation. Aftertransformation, Chinese sweetgum leaves could be induced different number of positive shootsstrained by different mannose and sucrose concentration. Higher PCR positiverate(82.0±15.7)and the largest positive shoots (7.7±3.8)were obtained with 15 g/l mannose and10 g/l sucrose in the medium.(3) Transgenic plants were obtained by using mannose selection system.Genetic transformation was confirmed by PCR and CPR (chlorophenol red assay), 134transgenic plants were obtained. PCR-Southern and RT-PCR analysis showed that AtNHX1gene was integreted into the genome of Chinese sweetgum, and could express normally intransgenic plants.