Trasfering Soybean Gene Into Hedysarum Mongolicum By Low Energy N~+ Implantation

Hedysarum mongolicum is one of the peculiar grazing in China.It is one of the good materials which can be used in checking winds and fixing drifting sand,it can be also used in improving the soil,changing the local conditions and improving the natural ecological environment.Because of the increase number of the livestock,herds again in the meadow in recent years,the natural Hedysarum mongolicum in China become fewer and fewer and it has already been disappeared even in some places of China.Utilizing genetic engineering to improve heredity of Hedysarum mongolicum has become an important practice meanings.Establishing an effective,steady system of tissue culture and transform system have become more important in molecular biological study and a key technology of genetic engineering of Hedysarum mongolicum in recent years.In this paper,we take the mature seeds of Hedysarum mongolicum as explant and establish the system of tissue culture,the factor of influencing induced rate of Hedysarum mongolicum callus has been studied preliminary.The experimental results show that when adopting MS basal medium,adding 2,4-D benefit to induce callus;adding 6-BA can improve callus quality obviously.Finally,the substrate of inducing mature embryo callus that adding 2,4-D(2mol/L) with 6-BA(1.5mg/L) for MS substrate has been established.This experiment contrapose to the resisting gene of kanamycin carried on recombinating plasmid pBI121/Gy3,study the influence of different consistency of antibiotic to increase rate of callus's weight, ensure the appropriate sieving consistency of kanamycin is 60mg/L and establish the effective filtration system.The effects of low energy N⁺ implantation on Hedysarum mongolicum mature seeds are also studied.The results show that ion beam energy has certain influence on survival rate of the seed.The curve of relation between the influence and survival rates of Hedysarum mongolicum dry seeds with differnece N⁺ implantation dosages are considered that the feasible transformation dosages are 800,900,1000×2.6×10¹³N⁺/cm²,of which the best transformation dosages are 900×2.6×10¹³ N⁺/cm²;by state the water lose rates and weight increase rate and determine the cell survival rates of callus with different implantation dose,the suitable transformation dosages are 100,150,200×2.6×10¹³N⁺/cm²,of which the best transformation dosages are 150×2.6×10¹³ N⁺/cm². When leading the experiment of transformation,the Hedysarum mongolicum dry seeds and callus is the receptor,and the riddling mark gene is GUS gene,the goal gene is pBI121/Gy3.About 100 seeds and 20 callus has been injected in each disposal group,adding kanamycin in inducing substrate to filtrate,and get the fastness callus after one month.The blue cells expressingβ-glucuronidase is observed at Hedysarum mongolicum dry seeds faced to ion beam and 13%transient expression is obstained.With the precondition that invitro tissue could survive after implantation,the study on transferring GUS gene into Hedysarum mongolicum callus is done,and the transformation efficiency is up to 2%.PCR analysis of the transformed plants show that the soybean gene have been integrated into the Hedysarum mongolicum genome.When studying on ion beam assistanted agrobacterium transformation Hedysarum mongolicum callus,in this experiment system,group that used agrobacterium transformation callus directly has no transformants.But in another groups that ion beam assitanted agrobacterium transformation Hedysarum mongolicum callus,we get transformants and transformation efficiency is 1%.