| Adventitious root is an important topic both on the theoretical aspect of plant organogenesis and on the practical side of plant propagation and excised tissues or organs in vitro regeneration. Factors affecting rhizogenesis were investigated, technical-platform of adventitious root was optimized and established, and anatomical and IAA immunolocalized characteristics in the process of rhizogenesis were studied with Poplar 741 in vitro in this research. The main results were as follow:1. A stable, efficient, and synchromic technical-platform of rhizogenesis was established. Leaves, stems, leaf discs and petioles from rooted plantlets and subcultured cultures of Poplar 741 were taken to compare their rooting difference, and the effect of IBA concentration on the rhizogenesis was also studied. The achieved results were that leaves from rooted plantlets were obviously better than other explants in aspects like time of rooting, rooting rate and synchronism in 1/2MS medium with 0.5mg/L IBA. The adventitious root started at the 6th day, and the rooting rate was up to 100% at the 8th day, and the average number of root was 3.7 in this system.2. TIBA application inhibited rhizogenesis of Poplar 741 leaves in vitro significantly, suggesting IAA polar transport plays an important role in the rhizogenesis. The rooting rate was 52.2% at the 8th day in the treatment of 5mg/L TIBA, decreased 47.8% compared with the control.The rooting rate was 5.6% at the 8th day in the treatment of 5mg/L TIBA, decreased 94.4% compared with the control. When TIBA increased to 20mg/L, the basal of petiole had no root and grew out callus. When the concentration of TIBA increased to 100mg/L, the leaves was hurt and the laminae get yellow. So the suitable concentration of TIBA to inhibit the adventitious root was 10mg/L.3. Anatomic result showed: root primordium was induced root primordium in the Poplar 741 leaves.The root primordium originated from the cambium cells.The process of adventitious root was: the cambium cells divided to form cell groups, the cell groups developed into root primordium and the primordium cells grew and developed to adventitioud root.4. The technique of immuno-gold localization of endogenous auxin was improved with embryo of walnut and poplar tender shoots. (1) considering the woody plants characteristics of perennial gowth, compound complexity, antigen hiding in the networks, urea together with trypsin was used to unmask the antigens of tissue sections. (2) the concentration of Triton X-100 in the buffer was improved to increase the permeability of cell membrane. (3) 2.5% BSA was added to the buffer to eliminate the background staining. (4) the reaction condition between antigen and antibody was at 37℃for 2h instead of 4℃overnight. The improved methods have characters of high sensitivity, strong speciality and clear background.5. The results of immuno-gold localization of IAA in the rhizogenesis showed that IAA was accumulated at the cambium during the induction stage. Application of 10mg/L TIBA delayed the accumulation of IAA. The details of the results were: little IAA was found in the basal of petiole before induction, the density of gold particles in cortex and vascular tissues were 4.8/100um2 and 5.1/100um2. At the beginning stage of induction, gold particles mainly distributed in vascular tissues and the density was 216.8/100um2. At the stage of root primordium formation, gold particles mainly distributed in the primordium and the density was 345.7/100um2. At the development stage of root, IAA mainly distributed in the root tip and stele, and the densitys were 210.5/100um2 and 30.1/100um2. At the 3th day after application TIBA, little IAA was found still in the basal of petiole, the density of gold particles in the cortex and vascular tissues were 6.9/100um2 and 8.7 /100um2. At the 6th day, gold particles maily distributed in the vascular tissues and the density was 150.2/100um2. At the 8th day, gold particles mainly distributed in the root primordium and the density was 300.5/100um2. At the 10th day, mainly distributed in the root tip and stele, and the density were 220.4/100um2 and 30.1/100um26 Subcellular localization of IAA in the rhizogenesis of Poplar 741 leaves was studied with immuno-gold microscope technique. Cytoplasm, cell wall, vacuole, plastid and golgi body were labeled with gold particles at the differentiation and primordium formation stages. It was not found that gole particles accumulate in the specific cell organ, indicating IAA regulated rhizogenesis was transported to the destination tissue from other tissues organs.
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