Establishment Of Agrobacterium-mediated Transformation System And The Transfer Of Chit42 And PhyB Genes Into Citrus

Citrus is the most widely grown fruit crop worldwide.Conventional breeding of citrus is limited in application due to nucellar embryos,heterozygosity and long juvenile period.The development of genetic transformation has provided new opportunities for plant improvement and made it possible to modify just one or two specific traits maintaining the original characteristics of the cultivar.In this thesis,factors related to the in vitro regeneration and Agrobacterium-mediated transformation with the internodal stem segments taken from seedlings of 'Xupu' and 'Bingtang' sweet orange(Citrus sinensis),'Xinnv' ponkan mandarin(C.reticulata) as well as the rootstock Poncirus trifoliate,and from matrue materials of 'Newhall' and 'Bingtang' sweet orange,'Xinnv' ponkan mandarin. chit42 and phyB genes were introduced to the above mentioned cultivars by Agrobacterium-mediated.Transgenic plants were detected and confirmed by PCR, southern blot and RT-PCR Analyses.Main morphological and physiological characteristics were observed and measured.1.Establishment of an efficient system for in vitro regeneration from seedling epicotyl explants of citrusSeedlings were taken from 'Xupu' and 'Bingtang' sweet orange(Citrus sinensis), 'Xinnv' ponkan mandarin(C.reticulata) as well as the rootstock Poncirus trifoliate, as explants and various media were tested for their regeneration.It was found that the effective bud induction medium was MT plus 3mg/L 6-BA and the shoot multiplication medium was MT containing 1.5g/L malt extracted and 0.5mg/L 6-BA. The highest rooting percentage was obtained on MT medium containing 0.5 mg/L IBA.This system of in vitro regeneration worked well for all the citrus cultivars tested.2.Genetic transformation of citrus with seedling epicotyl as explantsAgrobacterium-mediated transformation of citrus from seeding epicotyls with chit42 and phyB genes were carried out.The Agrobacterium-mediated transformation was optimized by the improvement of explants,co-culture and the determining the concentration of plant regulators in the medium.The resulted showed that the efficient system was the procedure composted of 30d old of sweet orange seedlings infected with Agrobacterium strain for 20min,transferred to co-culture medium(MT+3mg/L 6-BA) for 3 days and then on selective medium(MT+3mg/L 6-BA+100 mg/L Kan +500mg/L Cef) for 1 month.The regenerated buds were in vitro grafted to get the regenerated transformed plants.The induce ratio of resistant bud of sweet orange with chit42 gene reached 27.8%.Based on this system,chit42 and phyB genes were transferred into 'Xupu','Bingtang' sweet orange,'Xinnv' ponkan mandarin and Poncirus trifoliate rootstock,respectively.In total,obtain 16 transgenic clones and 235 transgenic plants.3.Transformation with citrus matrue internodal segments as explantInternodal segments were taken from adult plants of 'Newhall' and 'Bingtang' sweet orange as well as 'Xinnv' ponkan mandarin maintained in the greenhouse for transformation experiments.The results indicated that the contamination rate of the pretreated disinfection method was the lowest;about 1cm length of the internode stem segments gave the best effects;adventitious buds induction for 'Newhall' and 'Bingtang' oranges was the most effective on MS medium supplemented with 3mg/L 6-BA,but for 'Xinnv' ponkan mandarin was MS medium supplemented with 1 mg/L 6-BA and 0.5mg/L NAA.Agrobacterium-mediated transformation of matrue internodal segment was performed by infection with Agrobacterium strain for 20min,transferred to co-culture medium(MS+lmg/L 2.iP+2mg/L IAA+0.5mg/L 2,4-D) for 3 days and then on selective medium(MS+1mg/L 6-BA+30mg/L Kan+500mg/L Cef).After 1 month, adventitious buds formed and were then micro-grafted onto seedlings of Carrizo citrange grown in vitro and one month later re-grafted on greenhouse-grown rootstocks.In total,obtain 15 transgenic plants.Now the transgenic plants are growing well in the greenhouse.4.Molecular verification and biological observations of the phyB transformed Poncirus trifoliate plantsIn total 27 Poncirus trifoliate plants were regenerated and 5 transgenic clones were obtained by PCR analysis.RT-PCR analysis indicated that the transgene phyB regularly expressed in two transgenic clones phyB3 and phyB4.Morphological and physiological characteristics were observed on the two transgenic clones phyB3 and phyB4 in comparison with the untransformed clones obtained following the same regeneration procedure.The phyB transgenic plants show higher net photosynthesis rate than the control.All the regenerated transgenic plants,as compared with the untransformed clones,showed lower plant height and shorter internode length with larger leaf insertion angle than the control.5.Molecular analysis and disease resistance assay of the chit42 transgenic citrus plantTransgenic plants were detected by PCR and confirmed by southern blot. Southern blot results revealed that three and two copies were inserted to SR23 and SR24 lemon transgenic clones,one copy inserted to 'Xupu' sweet orange transgenic clones,respectively.RT-PCR analysis was performed and the results indicated the normal expression of the inserted gene.The transgenic clones of lemon were in vitro and in vivo tested for disease resistance to Citrus Anthracnose.The transgenic plants showed significantly higher resistance than the control plants by delaying the development of disease symptoms. Expression analysis of chit42 gene in transgenic lemon by semi-quantitative PCR indicated that the expression of chit42 gene was enhanced after 48h of infection.The transgenic plants grew normally in the greenhouse without any morphological differences in comparison to the control plants.