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Establishment of Tomato Roots in Vitro Dual Culturing System and Its Preliminary Identification of Signaling Molecule for Formation of Arbuscular Mycorrhizal Symbiosis

Author: WangJingJing
Tutor: SunShuBin;XuGuoHua
School: Nanjing Agricultural College
Course: Plant Nutrition
Keywords: arbuscular mycorrhiza tomato Agrobacterium rhizogenes Glomus intraradices dual culture HPLC
CLC: S641.2
Type: Master's thesis
Year: 2009
Downloads: 27
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Abstract

Abuscular mycorrhizal (AM) fungi form mutualistic, symbiotic associations with the roots of more than 80% of land plants, which facilitate plant mineral nutrient uptake, enhance its tolerance to environmental stress and plant diseases. The fungi contribute to maintenance of plant diversity and ecosystem. The establishment of function symbiosis between AM fungi and host plants involves the secondary metabolites in plant leading to the morphological integration of the symbionts. The switches in the fungi are triggered by host signals which induce changes in differential hyphal branching, appressorium formation, root colonization and arbuscular development. So it has significant meanings that investigation on the difference of exudates between Micro-Tom WT and Myc- m161 using hairy roots in vitro dual culturing system will conduce to find out the key signaling compounds in the AMF-plant interaction.In this study, we first use Agrobacterium rhizogenes A4 to induce tomato (Lycopersicon esculentum) Micro-Tom hairy roots, and establish an in vitro dual culturing system with Ri T-DNA transformed Micro-Tom roots and Glomus intraradices. Then using High Performance Liquid Chromatography (HPLC) to separate and prepare the key different compounds between the two exudates of Micro-Tom WT and m161, add the compounds to the dual culturing system to observe the effects. The main results are shown as follows:1. The cultural system in vitro of Micro-Tom roots and a highly stable Agrobacterium-mediated transformation system were established. The results showed that the hypocotyl co-culture method had the highest transformation efficiency. The optimum concentrations of carbenicillin to removing Agrobacterium rhizogenes A4 was 500 mg/L.The growth curve was surveyed, and reached the most quantity at 21d. The transformation of T-DNA from Ri plasmid to the root in vitro of Micro-Tom was confirmed by PCR analysis.2. An in vitro dual culturing system was established with Ri T-DNA transformed Micro-Tom roots and Glomus intraradices. The result indicated that multilevel branched hyphae were observed about 14 days, and some hypha tip became expanded, gradually produced some newborn spores at 17～21 days after infection. About 600～800 mature spores per dish were obtained after 3 months of dual culture. The harvested spores were capable of germinating and subsequently colonizing the host hairy roots immediately. The successful in vitro dual culturing of tomato hairy roots and mycorrhizal fungi provided the ideal experimental system for conducting the researches on physiological and molecular mechanism of tomato mycorrhiza.3. Collected the exudates of Micro-Tom WT and Myc- m161 using liquid culture method, measured and prepared the content differences between the two exudates using HPLC. The result indicated that some compounds in WT were more than m161 under the same condition.4. Added the prepared compounds to the dual culturing system to examine the effect in the AMF colonization. We observed the newborn hyphae and spores at 21d in m161 which added the prepared compounds just same as wild type. As contrast, the phenomenon was found out in the negative control at 41d. So, we speculated that the prepared compounds would be the key signaling compounds which lacked by the mutant m161 in the presymbiotic phase.

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CLC: > Agricultural Sciences > Gardening > Vegetable gardening > Solanaceous > Tomatoes ( tomatoes )
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