This method could help in the development of reniform nematode-resistant cotton varieties. Through the identification of resistant genotypes from other cotton species and the evaluation of breeding populations. The main advantage of this technique is that it provides a simple and nondestructive method for reniform nematode screening.
Begin by placing a ball of cotton in the bottom of one four centimeter diameter, 21 center height conical plastic pot per seed. And filling the pots with steam pasteurized soil mixture to approximately two centimeters from the top of the pot. Place a plastic stake into each pot designating the genotype to be planted and plant one seed of the appropriate corresponding cotton genotype into each pot.
Fill each pot with additional soil to cover the seed and place the pots in a growth chamber set to constant temperature of 28 degrees Celsius with a 16 hour fluorescent and incandescent lamp photo period. Then place water emitters into each pot and use an automatic watering system to water the pots twice daily. Seven days after planting, create a small depression in the soil next to each plant and add one milliliter of reniform nematode suspension into each depression.
After inoculation, pot water needs to be carefully controlled in order to prevent washing the nematodes from the root zone. 28 days after inoculation, use scissors to remove most of the fully expanded leaves from the plants before squeezing each pot and sliding the soil out into one hand to remove the plants. Gently agitate the root system in tap water in a 10 liter container to remove the soil from the roots, followed by a brief rinse in a container of clean tap water.
Use scissors to remove the cleaned root system from the plant approximately one centimeter below the soil line. Place the roots into a plastic 120 milliliter non-sterile disposable specimen container. Next, completely cover the root system with approximately 30 milliliters of red food coloring solution and place the specimen container in a microwave oven for heating of the staining solution until it begins to boil.
When the sample has cooled to room temperature, replace the red food coloring solution with 100 milliliters of tap water to remove any excess stain. Then place the cover on the specimen container and store the sample at four degree Celsius until root infection analysis. To recover the plants for seed production, place a ball of cotton in the bottom of a new conical plastic pot and partially fill the pot with peat moss potting medium.
Place one root system harvested vegetative shoot in the pot and firmly add potting medium to fill the pot. Place a new labeled plastic stake into each pot to designate the cotton genotype and place the tray of pots into a plastic container of water. Briefly water the plants to moisten the potting medium and place the pots in a growth chamber set to a constant temperature of 28 degree Celsius and a 16 hour photo period.
After approximately 30 days, partially fill one six liter plastic pot per plant with potting medium. Transfer each plant into a six liter pot, firmly adding potting medium to each pot as each seedling is planted. Then place the plants in a glass house and add water to moisten the potting medium.
To evaluate the R.reniformis root infection, remove a root sample from the specimen container. Use a stereo microscope to count the number of female nematodes attached to the root system under 20X magnification. A successful nematode root system infection can be influenced by several factors, including the viability of the nematode used for the inoculation and seasonal variation, as the nematodes are less active during the winter months.
Then place the root system on paper towels for approximately 10 minutes to remove the excess moisture. Weigh the root system to determine the fresh root weight. Variations in root growth are common between excisions.
These variations, as measured by fresh root weight, can also be observed between plants of the same genotype. Relatively fewer female reniform nematodes are able to establish a feeding site for the resistant cotton genotype compared to the susceptible genotype. The fresh root weight data can be used to calculate the number of female reniform nematodes per gram of root tissue for each genotype.
The number of female reniform nematodes per gram of root for resistant genotypes is generally less than 10, whereas susceptible genotypes typically have greater than 30 nematodes per gram of root. Here, a subset of data from a segregating F2 population that included 300 plants is shown. These ranges in variation for root growth and nematode infection of the root systems are commonly observed for nematode evaluations, illustrating the ability of this procedure to be used to screen a large number of plants in a single experiment, to minimize this variation, and to facilitate an accurate assessment of the genetics of resistance.
Following this procedure, the genetics of reniform nematode resistance can be determined to identify the DNA markers associated with resistance. These markers can then be used for marker-assisted breeding and for the transfer of nematode resistance to upland cotton. After its development, this technique provided a non-destructive method for allowing researchers to recover plants after nematode re-screening to aid in the breeding of resistant cotton varieties.
