Studies On The Transformation Of Succari With DefH9-iaaM Gene And TERF1 Gene

Citrus is the most important fruit crop in the world.Now its annual production in the world is 110 million metric tons,with a total acreage of 7.15 million hectares (ha) and an annual volume of international trade about 650 million dollars.China is recognized as one of the key original centers of citrus,with a long history of cultivation,plenty of natural resources and selected varieties for commercial use.In 2006,the planting area of citrus in China exceeded 1.7 million ha,which was 23%of the total acreage and ranked the first place in the world.The annual yield of citrus was nearly 18 million ton accepting the second biggest country for citrus production in the world.China has a long history of citrus breeding and has established the foundation of citrus industry,and the distance on technology application is widely accepted compared with those advanced countries of citrus production.In order to catch up with the international trade standard,the most important thing is to produce fruit of high quality and to satisfy the consumers.The criteria for evaluating the citrus cultivars are summarized as the following five main aspects:the appearance,taste,nutritive content,seeds number and easy-peeling by hand,et al.From the view of botany,the citrus is as a woody,low branching plant with a distinct juvenile stage,polyembryony,high genetic heterozygosity and arthenogenesis,and these characteristics have impeded the traditional breeding in citrus,such as the blindness in crossing and low efficiency for new cultivar development.With the development of biological technology,using transgenic technology to breed new variety is the best way to modify the genetic expressing or silencing.Transferring the foreign genes into mature explants will save us a lot of breeding time,and the gene expression in flower and fruit can be investigated much earlier than traditional did.Succari is a very popular sweet orange,almost acidless,and is extending quickly in the south of China,but with plenty of seeds.Liking other sweet oranges, Succari trees can be attacked by many biotic(bacteria,fungi,etc.) and abiotic stresses. The objective of this project is to establish the regeneration and transformation system of Succari orange and to transfer a parthenocarpy gene 'defH9-iaaM' and a transcription factor 'TERFI' gene into the genome by using seedlings and adult inter-nodal segments as explants.Hopefully to gain a new germplasm or breeding material,which carrying parthenocarpy gene and got a broad-spectrum resistance to diseases.The DefH9-iaaM gene consists of an iaaM gene and a DefH9 promotor.The transcription factors TERF1 gene as cloned from tomato by yeast-one-hybrid system. After the disease inoculation and morphological trait observation,we evaluated the modification capability of the wild plants by foreign gene.The main results are showed as follows:1.Establishing the regeneration system from Succari orange seedling sterms intemodal segments.The results indicated that 30-day-old seedlings were the most efficient explants,and the co-culture in darkness for 6 days gave much improvement for good regeneration.The optimum medium for adventitious buds regeneration was MS + 6-BA(5.0mg/L).And MS medium supplied with 6-BA1.0mg/L and NAA 0.1mg/L showed the highest bud multiplication ratio.The shoot tissues got rooted successfully after 20 days in 0.5mg/L NAA.The better way to get more adventitious buds is using the "two-step" method,which is to put the explants from the medium plus the auxin first,and then remove to medium with no hormones after 6 days.2.To set up an efficient regeneration system for mature sterms internodal segments.It shows that pretreatment disinfection method can raise significantly the survival rate to 80%.And the medium supplied with 6-BA 5 mg/L,2,4-D 2 mg/L and IAA 2mg/L could staminate the callus from cutting ends.And the callus induction rate reached up to 17.8%and 2.87 for the adventitious buds.The effect of three grafting methods on improving survival rate of adventitious buds was investigated,and the result showed that modified "⊥" way was the best one,which 78.94%of survival rate.3.Development of the transformation system and the transfer of DefH9-iaaM gene and TERF1 gene into Succari.The effects of transformed by different Agrobacterium strains was observed,and it seemed that EHA105 was better than LBA4404.And 15 min was the best infection time.The selection concentration of km is different between the seedlings and mature explants,and 100 mg/L for seedling explants and 30 mg/L for mature explants were justified.4.PCR results indicated that six regenerate plants of Succari were transgenic with the DefH9-iaaM gene,and the six transgenic plants showed evident increase in plant height,stem length,and leaf area.Through the PCR and RT-PCR analyses,it was showed that 7 regenerants were cooperated with the TERF1 gene.No.3,No.7 and No. 11 transgenic lines were verified by Southern blotting,and the results showed that one copy of the TERF1 gene were insert the plant genome.5.After inoculation with the citrus canker and anthracnose pathogen the transgenic plants of TERF1 gene,the results showed that the TERF1 gene can improve the resistance to both diseases.The transgenic plants could reduce citrus bacterial canker incidence rate,by 70%.The No.3 showed 0%of the incidence rate.When the transgenic leaves and control ones were both infected with the citrus anthracnose,all the leaves showed blots symptom of disease.The blots on transgenic leaves were smaller than that on the controls.Leaves of No.3 and No.7 plants did not have the disease blots,and the color of leaves also kept green.