Establishment Of Thellungiella Halophila Transformation By Agrobacterium-mediated

Soil salinity is a resource and ecology problem worldwide.According to statistics,there are 950 million hm² of land of all ldnds of salinization today,which is increasing.The state of salinization in China is serious too.Soil salinity harms social economics,especially agriculture.It is very important and pressing to solve it.Compared to traditional method,there are advantage such as lower costs,and protecting and environment to solve soil salinity on biotechnology,all of which,has a wide prospect.Thellungiella halophila,as well as Arabidopsis,belonging to Thellungiella of Cruciferae,is an annual herb.Thellungiella halophila is similar to Arabidopsis on a lower plant,shorter growth,an abundance of seeds,autocarpous,ect.,which is a close relative of Arabidopsis with over 90%homologous sequence on the cDNA level.So,it is convenience to use the genome sequence of Arabidopsis.In addition,as halophyte,low salt concentration can increase fresh weight,dry weight and photosynthetic speed of Thellungiella halophila,which can finish their history of life in the condition of 300mMNaC1 and exist in high salt environment(500mMNaCl).In addition,Thellungiella halophila can tolerate -15℃low temperature and drought.Recently,Thellungiella halophila was adopted as a model for salt tolerance research with the value of potential theory and practice on plant solt tolerance.Agrobacterium tumefaciens Ti transformation system is one of most current to studies with mechanism very clear,and a technique method that is the most mature transformation system.Heretofore,over 80%oftransgenic plants,which is close to 200 kinds are benefited by that.The transformation methods by Agrobacterium-mediated contain variety,among them,leaf disc transformation,whiCh is common as well as in dicotyledon.The purpose of this experiment lies in establishment of Thellungiella halophila transformation.In this experiment,it were researched the effect of different NaClO disinfection time on pollution rate of explants and different plant hormones and densities to effect callus induction,adventitions bud differentiation,rooting,perfecting tissue culture technology and building up efficiently regeneration system;Studying the influence on Thellungiella halophila by Agrobacterium-mediated with the way of AS increase,coculture time and selection pressure and eliminating Agrobacterium adhere to the explants for establishment efficiently of Agrobacterium-mediated transformation system to lay foundation.In this experiment,we used leafstalk as explants to establish the regeneration system through callus induction,adventitions bud differentiation,rooting and transplant.On this basic,transformation system of Thelhmgiella halophila was set up by preculture, coculture,selective culture and histochemical GUS assay.The main results were as fellow:(1)MS basal medium Supplemented with 2.5mgL^-1 6-benzyladenine(6-BA),0.1mgL^-1 naphthalene acetic acid(NAA) was the medium for callus induction and adventition bud differentiation of Thelhmgiella halophila.Adventition bud differentiation need 30d. Differentiation rent was 67%.The rooting medium was half strength MS basal medium. Rooting need 15d.6-benzyladenine(6-BA)+2,4-dichlorophenoxyacetec acid(2,4-D) could induce callus but not differentiate adventition bud.(2)Precultured for 3d on preculture medium supplemented with 2.5mgL^-1 6-benzyladenine(6-BA),0.1mgL^-1 naphthalene acetic acid(NAA).Bacterial suspension OD₆₀₀0.7,infection time 15min.Cocultured for 3d in dark on coculture medium supplemented with 2.5mgL^-1 6-benzyladenine(6-BA),0.1mgL^-1 naphthalene acetic acid (NAA),19.6mgL^-1 acetosyringone(AS).Coculture time less than 3d go against Agrobacterium fully invading,more than 3d harm exptants by Agrobacterium breeding in great quantities.Selection medium supplemented with 2.5mgL^-1 6-benzyladenine(6-BA), 300mgL^-1 cefotaxime,10mgL^-1 hygromycin.Rooting medium is half strength MS basal medium containing 300mgL^-1cefotaxime and 10mgL^-1hygromycin.(3)Selection culture for 4d,the explants were washed with sterile distilled water per three days and change the selection medium.It can efficiently reduce Agrobacterium breeding.(4)Eight weeks latet,hist0chemical GUS assay showed that transgene expressed in leaf with transformation frequency of 5%.