The overall goal of this procedure is to inoculate through needle injection, a USTA Lago maus cell suspension culture, and carry out a disease resistance reaction screening of maze tete and maze teosinte cross intres lines. This is accomplished by first selecting plant lines for inoculation and screening. Next, the seeds for experimental and control needle injection inoculation experiments are planted.
10 days later, the U made cell suspension is injected into the stem of each plant. Finally, the resistance reactions of the experimental and control plants are compared. Ultimately, results can be obtained that show a successful needle injection inoculation through visualizing the phenotype of the plant inoculated with you.Mais.
The main advantage of this technique is that it delivers the pathogen directly between the leaves. This eliminates the pathogen penetration issue. To begin select plant lines for inoculation and screening as shown here, two maze lines, five teosinte lines, and 40 maze Teosinte crosslines with uncharacterized resistance to U Mais were used for this study for experimental and control needle injection inoculation experiments.
Plant four seeds of each plant line in small flats by pushing the seeds about half an inch into the soil with a finger and lightly covering them with soil. Then place the plants in a growth chamber and water the seeds until soaked, ensuring that they remain under the soil After watering. Grow the plants with day and night environments of 28 and 20 degrees Celsius, and 14 and 10 hour light and dark cycles respectively.
And approximately 500 micromolar per square meter per second of photosynthetically active radiation at the top of the canopy. Maintain the relative humidity during the day and night at approximately 70%and 90%respectively, and keep all the plants in the same growth chamber to maintain a growth environment that is congruent across the experiment. Eight days after planting the seeds, use a sterile loop to streak huis from a frozen glycerol stock onto potato dextrose agar or PDA plates incubate at 30 degrees Celsius for two days and monitor it regularly to ensure it's growing well under a laminar flow hood.
Use a sterile toothpick to select a single colony for each strain and place them into three milliliter tubes of PD broth. Incubate the cultures at 30 degrees Celsius and 200 RPM for two days, checking periodically for growth. Next, measure the OD 600 of the cultures to ensure that they were grown to an OD of 1.0.
Using water, bring the cultures to a final concentration of 1 million cells per milliliter in a final volume of 30 milliliters. This concentration of pathogenic cell suspension consistently results in good infection of the plants. Then prior to inoculation, mix equal volumes of the two you made as strains.
Before filling a three milliliter syringe with the cell suspension, fill the control syringe with water to inject with minimal damage to the plant. Tissue attaches zero point 457 millimeter by 1.3 centimeter hypodermic needle to the end of each syringe. Remove the plants from the growth chamber and to inject at a 90 degree angle just above the soil line.
Carefully insert the needle of the U maus into the stem of an experimental plant until it is in the middle of the stem. Inject approximately 100 microliters of the cell suspension. It will push through the stem and be visible as it moves into the whirl of the plant.
Continue injecting additional experimental plants until the syringes empty. Then fill it with water to clean the needle of any plant tissue and refill the syringe until all plants are injected. Use the water syringe to inject the control plants in a similar manner.
When the injections are complete. Place the plants back into the growth chamber on a daily basis. Water only the soil, and check for pathogen development and plant resistance reactions.
Using a one to five resistance reaction rating, scale, score and record the resistance reactions for each plant 7, 10, 14, and 21 days post inoculation or DPI. The numerical values on the rating scale increase with increasing disease severity. For example, A1C one A or two resistance reaction indicates resistance.
A three, four, or five resistance reaction indicates susceptibility. Compare the resistance reactions of the experimental and control plants and select experimental plants with A1C one a or two resistance reaction rating. These plants are considered to be resistance to matis.
In this experiment, the majority of the plants inoculated humus, were susceptible to infection. Plants showed severe disease development demonstrated by stem and basal gall formation with black telio spores. Several plants were dead after inoculation due to the severity of the disease.
In contrast, the control plants were very clean and did not show pathogen development on any part of the plant, indicating that pathogen development on the experimental plants was due to the needle injection inoculation with U Mais three Mais Teosinte cross aggression lines that were resistant to U Mais were identified, and a successful inoculation was verified by minor sclerosis, cyan in production, or minor leaf gall formation. This technique can be done in a highly efficient manner with reproducibility. This helps in inoculation and phenotyping a large number of plants.
