Production of Arbuscular Mycorrhizal (AM) Fungal Inoculum and Phenotypic Evaluation of Rice and AM Symbiosis Under Saline Conditions

My research focuses on the formation and the function of arbuscular mycorrhizal symbiosis, aimed to understand how plants establish this relationship with AM fungi and how they benefit from it. We found that AM symbiosis enhances salt tolerance in rice by mediating gene expression. Additionally, tomatoes use a specific transcription factor to establish AM symbiosis, which regulates peptide hormone expression to promote lateral root growth. We aim to identify novel transcription factors crucial for AM symbiosis and further analyze the molecular mechanisms behind AM-enhanced salt tolerance and AM-induced lateral root formation.

[Narrator] To begin, wash sand thoroughly with tap water. Autoclave the sand to sterilize it. Add 2/3 of the sterilized sand to a pot. Add 1,000 spores of arbuscular mycorrhizal fungi, Rhizophagus irregularis, to the sand. Cover the sand and spores with a thin layer of sand. Then add 30 seeds of garlic chives to the pot, and cover them with an additional layer of sand. Place the pot in a growth chamber set to a 16-hour light and eight-hour dark cycle at 23.5° Celsius and 55% relative humidity. During the first week post-inoculation, cover the garlic chives with alumina paper to block light. From two weeks post-inoculation, fertilize the garlic chives twice weekly with 80 milliliters of half-strength Hoagland solution and once weekly with 80 milliliters of water. After 10 weeks, harvest the roots of garlic chives for trypan blue staining to assess fungal colonization. Store the dried sand inoculum in a plastic bag in the refrigerator at 4° Celsius. Transfer the inoculated garlic chive root pieces in a solution of 10% potassium hydroxide. Incubate the mixture at a temperature above 90° Celsius for 30 minutes. Remove the potassium hydroxide solution after incubation. Rinse the root pieces three times with double-distilled water to remove residual potassium hydroxide. Incubate the root pieces in 0.3 molar hydrochloric acid for 15 minutes to two hours. Remove the hydrochloric acid solution after incubation. Add 1 milliliter of 0.1% trypan blue solution to the root pieces. Then incubate them at a temperature above 90° Celsius for eight minutes. Wash the stained root pieces with 50% acidic glycerol. Then transfer 10 root pieces onto slides. Add a drop of 50% acidic glycerol. Seal the cover slips on the slides with nail polish to prevent leakage. Examine 10 fields of view for each root under a microscope to record fungal structures. Remove the holes from the rice seeds manually. Immerse them in 70% ethanol for four minutes and 30 seconds to sterilize it. Remove the 70% ethanol solution from the tubes. Then add 3% bleach into the tube and shake for 30 minutes. Remove the bleach solution and wash the seeds with sterile double-distilled water three to four times inside a laminar flow hood. Grow the sterilized rice seeds on half-strength Murashige-Skoog medium containing 0.8% agar. Incubate the seeds at 30° Celsius in the dark for five days. Transfer the rice seedlings to growth conditions with a 12-hour light and dark cycle at 28 to 30° Celsius and 70% relative humidity for two days. Then transfer the rice seedlings into plastic tubes containing sterilized sand. Do not add any inoculum to one tube labeled "mock." Then add 5 milliliters of sand inoculum containing spores of Rhizophagus irregularis to another tube labeled "test." Water the rice plants with double-distilled water every day for the first week after inoculation. Fertilize the plants every second day with half-strength Hoagland solution containing 25 micromoles of potassium dihydrogen phosphate. At five weeks post-inoculation, treat one batch with 150 millimoles of sodium chloride. Water the plants with half-strength Hoagland solution containing potassium dihydrogen phosphate, sodium chloride, and with just plain water on different days of the week. Harvest the plants at eight weeks post-inoculation, and measure their fresh weight. Place the plants in a 70° Celsius oven for two days to measure their dry weight. Use trypan blue staining to analyze fungal colonization levels. At 10 weeks post-inoculation, fungal structures, such as vesicles and spores characteristic of late-stage arbuscular mycorrhizal symbiosis, were observed in garlic chive roots, with total fungal colonization levels of 80%. At eight weeks post-inoculation, rice roots colonized using sand inoculum showed vesicles and spores, with intraradical hyphae at 91%, arbuscule at 82%, vesicle at 95%, extraradical hyphae at 46%, spores at 2%, and total colonization at 93%. Under salt stress conditions, mycorrhizal rice plants exhibited fewer wilted blade tips than mock plants. Under non-saline conditions, mycorrhizal rice plants showed higher shoot biomass than mock ones. Salt stress resulted in severely reduced shoot biomass of mock plants relative to mycorrhizal plants. Extraradical hyphae levels increased under salt stress, while other fungal structures remained unaffected, indicating that salt stress had a mild impact on AM symbiosis.