In vivo and In vitro Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes

The protocol describes infection of Solanum tuberosum roots with plant parasitic nematodes under in vivo greenhouse conditions and potato in vitro transgenic roots for histochemical analysis of root structure through optical microscopy.

Research on parasitic nematode infections in crops is influenced by the variability of environmental conditions, which can significantly impact experimental outcomes and data reproducibility.

We have developed in vitro cultures of potato transgenic roots with plant parasitic nematodes as a reliable alternative that occupies less space, requires less time to obtain, and it's free from contamination or from host genetic variability.

Tracking the temporal progression of nematode infection in crop roots remains challenging. Differential staining techniques provide a reliable method for identifying infection sites and distinguishing nematode life stages with precision.

Compared to greenhouse trials, potato heavy roots support a continuous and control system for providing all stage of nematode development, independent of seasonal or climatic variations.

So the protocol described offers several promising future applications. For example, it enables detailed studies into the molecular and cellular mechanisms of providing insights into how plant parasitic nematodes infect and manipulate hosts.

[Narrator] To begin, wash a carrot under running tap water and then with a common detergent solution to remove debris. Once dried with paper towel, insert a sterilized metal skewer into the top of the carrot, about one to two centimeters inwards. With a wash bottle fitted with a nozzle, wet the carrot with 96% ethanol. Blot the bottom tip of the carrot on a sterilized filter paper. Then carefully pass it through a flame. With a sterile peeler, peel the carrot from the top down and repeat flaming. After discarding the top and bottom sections, place the middle section in a sterile Petri dish. Using a sterile blade and tweezers, cut one centimeter thick sections. Transfer the sections into sterile Petri dishes. After sealing the plate, keep it under UV light for 60 minutes on each side for sterilizing the carrot discs. Incubate the carrot discs at 25 degrees Celsius in darkness for one to two weeks. Next, with a sterile blade, make an X shaped incision at the center of the carrot disc, cutting only halfway deep. Pipette 50 microliters of root lesion nematode suspension containing at least 50 mixed life stages into the incision. After sealing the Petri dish, incubate it at 25 degrees Celsius in darkness for up to three months. To extract the root lesion nematodes, transfer the carrot discs with visible necrosis to a 75 micrometer mesh sieve with eight centimeter diameter placed in a sterile glass bowl. Next, pour antibiotic solution over the sieve until discs are covered. After overnight incubation in dark, use a sterilized pipette to transfer the nematodes from the bottom of the bowl to a sterilized glass staining block. Pipette one milliliter of antibiotic solution into the staining block and let the nematodes settle for 30 to 40 minutes. Pipette out the used antibiotic solution and repeat the washing process four to five times. Use the root lesion nematode suspension immediately, or store at 11 degrees Celsius. Select potato tubers of the same size and discard any with holes, bruises, or soft sections. Fill five liter pots with a one-to-one mixture of autoclave soil and sand and mix in 22.5 grams of slow release NPK fertilizer. Then sow the potatoes at a depth of nine centimeters below the soil surface. Place the pots in a greenhouse under 50 to 70% humidity. Water frequently to maintain soil moisture at 70% of maximum water holding capacity, avoiding temperature extremes. Once potato plants have emerged, create evenly distributed four to six holes around each plant to see depth. Pipette eight milliliters of suspension containing 30,000 living mixed life stage root lesion nematodes into the holes. Then, cover the holes with soil mixture. For control pots and pots containing root lesion nematodes, withhold watering on the day of inoculation. After two months of culture, uproot the potato plants and separate the shoots and roots for weighing. Carefully wash the root system. Examine the roots for RLN attack sites using appropriate staining techniques. Place washed and sterilized potato tubers in a container. Cover the tubers with a 25% commercial bleach solution. Close the container and mix for 15 minutes. Once the bleach is disposed, rinse the tubers three times with sterilized tap water. Next, in a flow hood, immerse the tubers in 80% ethanol solution for 15 minutes with vigorous agitation. After pipetting out the ethanol, rinse three times with sterilized tap water. With a sterile scalpel, remove the peripheral portions of the tubers. Section the inner central piece into 0.5 centimeter thick segments. To inoculate the sections, first mix one milliliter of Rhizobium rhizogenes suspension with nine milliliters of SH medium. Dip the tip of a sterile scalpel into the diluted suspension and wound the surface of the potato segments five times. Once the segments are dried, place them on semi-solid SH medium and incubate at 25 degrees Celsius for three days in the dark to facilitate plasmid transfection. At the end of the third day, transfer the infected segments to plates containing semi-solid SH medium supplemented with antibiotics. After three months, use sterile tweezers to gather a one gram cluster of transgenic roots. Transfer the roots to the center of plate with fresh semi-solid antibiotic-free SH medium. To collect the nematode egg masses, obtain the root galls. Use a pair of sterile ultra fine point tweezers to carefully extract egg masses under a binocular stereo microscope set to 20 x magnification. Place the egg masses in a covered Petri dish containing five milliliters of sterile tap water and let them hatch for 48 hours. Next, in a flow hood, pipette five milliliters of the J2 suspension containing 100 nematodes per milliliter onto a 20 micrometer mesh sieve. After washing with sterile tap water, immerse the bottom half of the sieve containing J2 nematodes in a 20% hydrogen peroxide solution, and mix in circular motion for 15 minutes. Dispense sterile tap water through the sieve over the nematodes, and repeat the washing process two times. Tilt the sieve so that the nematodes collect at the border during the final wash. Then, pipette one milliliter of sterile ultrapure water from the sieve border to recover the nematodes. In a flow hood, subculture a one gram clump of potato transgenic roots onto SH plates with 100 sterile nematodes. Monitor the co-culture regularly under an inverted microscope at 100 x magnification. When egg masses become visible, subculture the roots to a new SH medium plate. The use of carrot discs resulted in an average 100 times increase in nematode populations within three months. Potato plants showed no visible symptoms under low root lesion nematode population numbers. Several life stages of Pratylenchus penetrans were observed in the root cortex after staining with acid fuchsin, indicating penetration of the tissue and the associated necrotic lesions were clearly visible in infected areas. Development of potato transgenic roots showed initial cell mass growth along scalpel induced wounds in the potato tuber section, followed by the emergence of transgenic roots. Sustained growth of the roots was observed in the culture medium and root clumps were successfully transferred to fresh culture medium for continued growth. Potato transgenic root cultures were successfully infected with Meloidogyne chitwoodi second-stage juveniles to establish plant nematode co-cultures. Root galls containing adult females and egg masses were observed in the infected cultures. Gall tissues formed by Meloidogyne chitwoodi were visible after infection of transgenic potato roots with distinct stages of nematode development and reproduction identifiable, including the formation of egg masses and eggs.