Evaluation Of Transgenic Citrus With MdSPDS1 Gene On The Resistance To Temperature Stress

Citrus is an economically important world fruit. However, the production of citrus islimited because of heavy losses due to biotic and abiotic stress (disease) such as cold andfreezing damage; high temperature injury, etc.. Although great efforts have been madethrough traditional breeding methods, citrus breeders have limited success owing tomany problems, i. e., nucellar polyembryony, high heterozygosity, inbreeding depressionand long juvenile period. Genetic transformation, which opened up new avenues todevelop citrus cultivars with new expected agronomic traits, is an alternative pathwayused by breeders to overcome these problems.In this study the transgenic calli of Murcutt tangor monoclonal cell line andtransformed potted plants of oval Kumquat with MdSPDS1 gene were used as materialsto investigate their physiological index under temperature stress in order to evaluate thepotential utilization future of MdSPDS 1 gene in citrus genetic improving. The mainresults are as follows:1. Murcutt tangor monoclonal cell line calli were separated by low density culture ofprotoplast from kanamycin resistant callus, three of PCR-positive clones were selectedfor further analysis. Real-Time RT-PCR showed that transgenic cell line dispayed higherexpression level of SPDS 1 gene than control callus.2. To survey the stress tolerance oftransgenic cell line, growth rate andphysiological index were examined. The results showed transgenic calli exihibited highergrowth rate with higher content of free polyamine (spd+spm)/put proportion undertemperature stress than the controle. The level of protein content and SOD activity intransgenic calli was up-expressed while MDA content remained lower level comparedwith untransgenic calli. These results indicated the transgenic calli had highertemperature tolerance than un-transgenic maternal callus. 3.7 potted PCR-positive plants of oval Kumquat were confirmed to be transformedby southern blot analysis and Real-Time RT-PCR. Among transgenic plants twotransgenic clones had higher relative expression of SPDS 1 than control plant.4. Under temperature treatment (4℃, 6h or 45℃, 6h), transgenic plants had higher(spd+spm)/put proportion, SOD activity and soluble protein content than untransformedplant while MDA content climbed less. The results indicated transformation ofMdSPDS 1 enhanced the tolerance of transgenic plants to temperature stress.To sum up, transformation of MdSPDS1 gene into citrus had effect to improve the(spd+spm)/put proportion and enhanced the resistance of transformants to temperaturestress.