Molecular Analysis, Morphological Evaluation And Propagation Of The Transgenic Citrange With Rol ABC Genes

For citrus, unlike other fruit tree species, there are no useful dwarfing rootstocks available and the existing ones have serious limitations in their uses because of their low adaptability to different environmental conditions or scions incompatibility. For this reason, obtaining dwarfing genotypes is one of the most important goals in citrus genetic improvement. With the aim of modifying the growth habit of an important rootstock, a research program was set up to introduce rolABC genes from Agrobacterium rhizogenes into the genome of Troyer citrange [C. sinensis (L.) Osb x Poncirus trifoliata (L.) Raf.]. And in this papper ,Molecular Analiysis and detailed observation of morphological and physiological characteristics were carried out on the grafted transgenic clones (B, D, E), The main study results were as follows: 1. DNA was extract by modified CTAB method from the leaves of the rol ABC transgenic clones (B, D, E) and the control citrange. the quality of DNA samples was suitable for PCR. Using special primers of rol A,B,C and npt II genes, PCR analysis indicated that the rol A, B, C genes were integrated into the citrange genome.2.RNA was islolated by Trizol from the leaves of the rol ABC transgenic clones (B, D, E) and the control citrange and treated with Dnase I .The expression of the transgenes npt II and rol C was detected by using RT-PCR; while the transgenes rol A and B did not show any RT-PCR product, and it was not clear for the factors related to their expression.3. Detailed observation of morphological and physiological characteristics was performed on the grafted transgenic clones (B, D, E). In comparison with the control plants, the rol transgenic plants showed evident dwarfing characters with about half of the control in plant height, shorter internodal length, weaker apical dominance with more lateral branches, smaller leaf area, and higher net photosynthesis rate. Higher contents of the endogenous GA3, IAA and zeatin were found in the middle and basal parts of the transgenic shoots than the control ones, this could be one of the factors responsible for the above described morphological modifications.4. In vitro propagation made it possible to speed up the multiplication of the rol ABC transgenic citrange plants, so as to quickly evaluate this new rootstock type. Using budsticks from the transgenic clones (B, D, E) and the control citrange as explants, various culture media containing various plant growth regulators were tested for bud burst, shoot multiplication and rooting. The results indicated that MS medium containing 1 mg·L^-1 6-BA resulted in fastest bud bursting, MS added with 6-BA 0.5...