The Characterization Of The Transgenic Malus Robusta With LeIRT2

Apple is one of the most important fruit trees with a total acreage of about 5697.8 khm2 in the world in 2002, of which 43.9% is planted in China (FAO, 2002.). Most apple trees in China are grown on calcareous soils and often show chlorosis due to iron deficiency. This has been a difficult-to-control physiological disorder with many important apple cultivars and caused significant economic losses in commercial orchards in China. Unfortunately, the widespread problem of iron chlorosis of feuit trees remains poorly understood and the results of the methods its correction are not satisfactory, and they sometimes cause serious envirnmental problems as well. It usually takes many years to genetically improve the horticultural traits of perennial fruit trees through conventional breeding, because these trees have a long juvenile period and a highly heterozygous genome. In this case, genetic engineering, e.g., plant transformation, can play a significant role in facilitating fruit tree breeding.Malus robusta Rehd. is a rootstock that widely used in China for high-yield fruit production. They are resistant to many diseases and tolerant to stress conditions except iron-deficiency. In this study, the transgenic Balenghaitang and Gexi No.l tobacco (Nicotiana tabacum cv.Gexi No.l) lines with tomato LeIRT2 gene were identified with PCR, PCR-Southern, Southern and RT-PCR techniques. Hydroponic experiment was carried out to determine the iron-deficiency tolerance of the transgenic plants. The grafting compatibilities between transgenic Malus robusta and the master apple(M pumila) cultivar were early tested by in vitro micrograft techniques. The main results are presented as follows:1. A total of 19 putative transgenic Malus robusta lines with the tomato iron transporter gene (LeIRTI) via selecting of GUS and km were obtained, 11 of which were verified by PCR amplification to carry a fragment of the transgene. Among them, nine were confirmed to carry the transgene by Southern blot analysis with one to three copies of the transgene integrated into the plant genome. The half fix quantify RT-PCR were carried outto identify transcription of LeIRT2 gene, six carrying one copy of the transgene were successful transcript.2. Nine transgenic plants of Malus robusta were confirmed by Southern blot analysis. The results indicated that one to three copies of the transgene were integrated into the plant genomes respectively, then they were hydroponically planted to test their efficiency to iron-deficiency, and the tolerance was only found in the transgenic T4 and T5 with single copy. Their leaf chlorophyll content and net photosynthetic rate were much higher than those of control, which showed severe chlorosis under the same condition. The rate of root H+ excretion and ferric-chelate reductase activity increased in transgenic lines and were substantially higher than those of control. Scanning photos of root absorption region and root hair by electronic microscope in transgenic lines and non-transgenic plants were different under same iron treatment.3. The Fe Mn, Zn content of leaves and roots of the transgenic lines and non-transgenic plants under the hydroponic experiment in iron-deficient full-strength solution medium with NaFeEDTA 1.00 X10~-6 mol/L was determined. The total Fe content of the transgenic ling roots and the activated Fe content of the transgenic leaves were notably increased. The Zn content of the transgenic line leaves and roots were notably increased too, but the Mn content was failed to increase.4. Using LeIRT2 gene from plasmid as the probe, the expression of LeIRT2 in the transgenic malus robusta during iron deficiency was investigated with tissue printing mRNA Northern hybridization. Strong hybridization signals were observed in the leaves and roots of the transgenic malus robusta which had been exposed to iron stress for 5 days.5. The grafting compatibilities between transgenic Malus robusta and apple (M pumila) cultivar Fuji, Gala and Stark Delicious were early tested by in vitro micrograft techniques. The resu...