The Genetic Transformation Of Malus Robusta Rhed. With RolC Gene And Its Expression

Malus robusta Rehd., one of the most commonly used standard apple rootstoc(?) inChina. It is adapt to a wide range of soil conditions and high level tolerance to droght,cold and saline-alkaline and compatible with most apple varieties, but it is too vigoro(?) tobe used for commercial purposes. For this reason, it would be of importance to introucegene to make Malus robusta Rehd. dwarf. The rolC gene, originated from theAgrobactrium rhizogenes, is believed to modulate the synthesis or activities of plant-growthregulators. The product of rolC induces phenotypic alteration in rolC-transgenic pant,including dwarfing of plant height, shortened internodes, smaller leaves and reduced aicaldominance. Therefore, we introduced the rolC gene into the apple rootstock Malus robsaRehd. and attempted to get a new type of apple dwarf rootstock cultivars.In this study, Malus robusta Rehd. was transformated with rolC gene by S(?)AT(Sonication assisted Agrobacterium-mediated transformation), sonication drecttransformation method. Molecular detections, such as PCR, Southern blotting, RT-PCR,Northern blotting, were used to detect the transgenic plants got from SAAT, sonicationdirect transformation method and Agrobacterium-mediated transformation method.Furthermore, we study the expression characterization and stability of the 3 clones got fromAgrobacterium-mediated transformation method, the research results as following:1. Malus robusta Rehd. was transformated with rolC gene from Agrobacteriumtumefacien through Sonication assisted Agrobacterium-mediated transformation, thefactors which influenced the transformation efficiency, such as sonication treatment time,opportunity and the As concentration in Agrobacterium suspension, were all investigatedvia GUS staining instant expression. The results demonstrated that the leaves were treatedfor 30 seconds with 100w power, after soaked in Agrobacterium suspension that OD₆₀₀ is0.6 containing As 75mg.L^-1 for 2 minutes, and then soaked for 2 minutes sequentially,then been placed in regeneration medium for 3 days co-culture. This treatment can get thehighest gus transient expression 81.5%. 683 leaves were transferred under the optimum##原图像不清晰 treatment condition, then 138 resistant callus and 15 resistant buds were obtained(transformation rate 2.2%). And of them 12 were verified to be transformants by GUSstaining.2. Malus robusta Rehd. was transformed with rolC gene by sonication directtransformation method. The factors which influenced the transformation efficiency, such assonication treatment time, power and the DMSO concentration in transformation buffer,were all investigated via GUS staining instant expression. The results demonstrated that theoptimum treatment is 80w, 20 minutes and 5% DMSO. 486 leaves were transferred underthe optimum treatment condition, then 237 resistant callus and 9 resistant buds wereobtained (transformation rate 1.85%). And of them 8 were verified to be transformed byGUS staining.3. 21 clones of rolC-transgenic Malus robusta Rehd. having passed the gusexamination got by SAAT, sonication direct transformation method andAgrobacterium-mediated transformation method respectively, were used as materials forfurther research. PCR, Southern blotting, RT-PCR and Northern blotting were made toidentify their integrated copy numbers and expression at transcription level.The results of determination of the rolC-transgenic plants got from SAATdemonstrated that rolC gene had been integrated in the genome of the 12 clones ofrolC-transgenic Malus robusta Rehd, 7 of the12 transformed clones had a single copy, 3 of12 transformed clones had 2 copies, 1 of 12 transformed clones had 3 copies, 1 of 12transformed clones had 4 copies, 10 of 12 transformed clones express at transcription level.The results of determination of the rolC-transgenic plants got from sonication directtransformation method demonstrated that rolC gene had been integrated in the genome ofthe 8 clones of rolC-transgenic Malus robusta Rehd, 2 of the8 transformed clones had 2copies,3 of 8 transformed clones had 3 copies, 2of 8transformed clones had 4copies,1 of8transformed clones had 6 copies,4 of 8 transformed clones express at transcription level.The results of determination of the rolC-transgenic plants got fromAgrobacterium-mediated transformation method demonstrated that rolC gene had beenintegrated in the genome of the three clones of rolC-transgenic Malus robusta Rehd, two ofthe three transformed clones had a single copy and another had double copies of the rolCgene integrated different integrated copy numbers have influence on the expression of eachrolC-transgenic clones, the clone with double rolC copies is less than the other two cloneswith a single rolC copy number on expression abundance at transcription level. 4 The biologic characteristics of the 3 clones got from Agrobacterium-mediatedtransformation method, such as proliferation, regeneration of leaf and rooting, wereresearched on the media containing different kinds and concentration plant hormone. Theresults demonstrated that: (1) The three rolC-transgenic Malus robusta Rehd clones all needsignificant lower than the control on the propagation of shoot, rooting and regeneration ofleave. The clones with one rolC gene copy were significant lower than the clone with tworolC copies. (2) The root numbers of the three rolC-transgenic Malus robusta Rehd clonesare all significant more than the control; the clones with one rolC gene copy weresignificant more than the clone with two rolC copies. The root length of the control wassignificant longer than the three rolC-transgenic Malus robusta Rehd clones; The cloneswith one rolC gene copy were significant shorter than the clone with two rolC gene copies.The root diam have no difference between the control and the transgenic clones.5. The shoot tip, leave and root of the three rolC-trausgenic Malus robusta Rehdclones were used as the material for determining the content of endogenousCTKs(including iPAs and ZR), IAA, GA₁₊₃, and ABA. It was found that the rolC genechanged the endogenous content, but not changed the distribution. The endogenous ofCTKs, GA₁₊₃, and ABA of different clones' different position three rolC-transgenic Malusrobusta Rehd. clones are all significance increased; IAA of the root of the rolC-transgenicMalus robusta Rehd. is significance increased, but IAA of the shoot tip and the leaves aresignificance decreased.6. The morphologic characteristics, such as plant height, the number of nodes, thelength of nodes and leaf area, were researched after the acclimated in greenhouse for 4months. The three rolC-transgenic Malus robusta Rehd clones all showed the characteristicrolC phenotype, such as dwarf shoot, shorter internodes, smaller leave area, shorter than thecontrol. The clones with one rolC gene copy were significant lower than the clone with tworolC copies. The rolC gene has been integrated into the genome of Malus robusta Rehd.and has expressed at the level of transcription. The results of the expression of rolC alterthe content of endogenous hormone and the morphology of Malus robusta Rehd. Thenumbers of transgene play an important role at the expression of rolC gene. Paraffinsectioning with optical microscopy was adopted to investigate the leaves and stemanatomical structures in rolC-transgenic Malus robusta Rehd. and the untransformedcontrol plants. The results demonstrated that stomatal dentisty in leaf lower epiderm of thecontrol was higher than the 3 rolC-transgenic Malus robusta Rehd. clones, cell number in leaf lower epiderm and Number of upper epiderm cell of the control were lower than the 3rolC-transgenic Malus robusta Rehd. clones and stomatal length and stomatal width of thecontrol had no difference with the 3 rolC-transgenic Malus robusta Rehd. clones.Palisade/spongy ratios in rolC-transgenic Malus robusta Rehd. leaves were 1.39-1.48 times(from 1.18 to 1.26) of that in untransformed plants(only 0.85),The rate of wood/bark,average trachea density, average trachea area and trachea total area/xylem area ofrolC-transgenic Malus robusta Rehd. were all lower than the control.7. We research the inheritance and expression stability of ralc gene in the process ofvegetative propagation of rolC-transgenic Malus robusta Rehd. using the first to the thirdsubculture shoots and regeneration shoots from leaves which were get without exotericresistance screen as materials. The PCR and Southern blot were used to research theinherence stability of rolC gene and RT-PCR was used to research the expression stability.Simultaneity, we also study genetic properties including the rooting ability andmultiplication ability. The results demonstrated that rolC gene not only can inherit but alsocan express stability in the process of vegetative propagation of rolC-transgenic Malusrobusta Rehd.. The results of research about the rooting ability and multiplication abilityalso demonstrated that both the subculture shoots and regeneration shoots all can get stablegenetic properties.8. Using the shoots in vitro as material, we research the expression of rolC gene inrolC-transgenic Malus robusta Rehd. under the stress of salt and lower temperature. Theresults show that rolC gene could express stably under both salt stress and lowertemperature stress. Furthermore, along with the treatment time increasing, the expressionabundance also increased.