Studies On High Frequence Regeneration System And Genetic Transformation By Agrobacterium In Dichondra Repens Forst

Creeping Dichondra(Dichondra repens Forst)is part of the Scammony perennial herbage plant,and may be considered as a kind of excellent turfgrass and ground cover plants because of its unparalleled specialties.But in the course of using,there are some shortages,such as leaf is easy to turn scorch under the low-temperature in winter;it is susceptible to Septori asp;leaf that has delicate and high succus content is grave encroached by sticky insect;it is prone to damaged by weed,and so on.This experiment used cotyledon of Dichondra repens Forst as experimental material,and systemically researched the methods of tissue culture,and debated the effect of combinations of different plant growth regulators and concentration on callus induction and differentiation,finally established high frequence regeneration system of Dichondra repens Forst.At the same time,genetic transformation was studied by using cotyledon co-cultivation with Agrobacterium,firstly established transformation system,and lay the foundation for mutation breeding,cell engineering and genetic engineering.The results are as following:1.Regeneration system of cotyledonThis experiment used cotyledon of Dichondra repens Forst as experimental material,and systemic researched the methods of tissue culture,and debated the effect of combinations of different plant growth regulators and concentration on callus induction and differentiation.The result indicated that the culture medium contenting 2,4-D,.NAA,IAA and 6-BA had no significant effect on callus induction,the range of induction rate is 80-100%;but the quality of callus was quite different,the best induction medium was 5.0mg/L6-BA+0.1mg/L NAA and 0.5 mg/L IAA+1.5 mg/L6-BA;the induction rate reached 82.61%;AgNO₃ was an important factor in callus differentiation,the best differentiation medium was 0.1 mg/LNAA+3.0 mg/L6-BA+2.0 mg/L AgNO₃, the rate of differentiation was 76%;the best rooting medium was 1/2MS+0.5mg/IBA,rate was 100 %.2.A tumefaciences-mediated transformation system of cotyledonit was studied that several factors affected genetic transformation of Creeping Dichondra (Dichondra repens Forst),and finally established genetic transformation system.Following the experiment results,it was confirmed the optimal transformation condition:cotyledon segments were precultured on MS+0.1 mg/L NAA+5.0 mg/L 6-BA for 2～3d.The A.tumefaceins cells which OD₆00 comes up to,0.6～0.8 or so were collected by centrifugation and resuspended in same volume of liquid MS containing 100μmol/L AS,then it was made the OD₆00 came up to 0.2～0.3 or so.The cotyledon segments were transferred into A.tumefaceins suspension as described above for 3～5min.After the A.tumefaceins cells on the surface of cotyledon segments were removed,the explants were co-cultured on MS+0.1mg/LNAA+5.0mg/L6-BA+100μmol/LAS with a filter paper for 3～4d for whole day illumination.When the co-culture was over,the explants were washed in liquid MS containing 200mg/L Cef for 2 hours.After removing the liquid on the surface of the explants,the explants were transferred to MS+0.1mg/LNAA+5.0mg/L6-BA+30mg/LCef to de-culture for 2～3d.After that,the explants were transferred to MS+0.1mg/LNAA+5.0mg/L6-BA+Smg/LKm+30mg/LCef to induce resistant calli.The explants were transferred to MS+0.1 mg/LNAA+3.0mg/L6-BA+2.0mg/LAgNO₃+10mg/LKm+30mg/LCef when the shoots formed.Regenerated shoots were inoculated into 1/2MS+0.5mg/LIBA+10mg/LKm for rooting selection.Using the optimized transformation system,gene were introduced into Dichondra repens Forst,and 2 Kan-resistant plantlets were obtained.