Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein. Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.
Here we present a simple kernel screening assay to analyze Aspergillus flavus growth and Aflatoxin production in maize kernels expressing an antifungal protein. Using a green fluorescent protein expressing Aspergillus flavus strain, we monitor the infection and spread of the fungus in mature kernels in real-time. This simple assay provides a reliable means of evaluating the maize kernel's ability to resist Aspergillus flavus infection and Aflatoxin production.
Results from this lab assay performed under controlled conditions on mature kernels correlates well with results from infection under field conditions. Demonstrating the procedure will be Dr.Rajtilak Majumdar, a postdoc from our laboratory, along with Christine Sickler and David Ambrosio, biological science technicians also from our lab. To begin, glue 20 two mm snap caps in a Petri dish to create KSA caps.
Let the glue dry for 48 hours before using the caps. In a biological safety cabinet spray a square bioassay tray with 70%ethanol in water. Let the tray air dry.
Add sterile chromatography paper to the dry tray. Spray nine of the KSA caps with 70%ethanol and let them air dry. Then place the caps on the bioassay tray.
For each transgenic maize line being tested, select 20 undamaged kernels and place them into a 50 ml centrifuge tube. Using a tabletop stereo microscope select only undamaged kernels for the assay. Damaged kernels are more susceptible for Aspergillus attack and produce more Aflatoxin, thereby skewing the results and not representing the true antifungal or antimicrobial traits of the variety.
Add 70%ethanol to each tube. Let the kernels sterilize for four minutes while occasionally gently shaking them. After this gently pour off the ethanol and gently rinse the kernels in sterile deionized water three times.
Obtain a six day culture of Aspergillus flavus stain 70 expressing GFP grown on V8 medium. Add 20 ml of sterile 0.02%Triton X-100 in deionized water. And use a sterile loop to scrape off the spores.
Pipette off the inoculum and transfer it to a 300 ml sterile beaker. Prepare a five-fold dilution with 0.02%Triton X-100 and use a hemocytometer to perform a spore count. Dilute again if necessary to obtain 100 ml with a concentration of four million spores per ml.
Pour off the inoculum into an empty beaker. Place the kernels into a sterile 300 ml beaker with a stir bar and stir for three minutes. Using forceps transfer the kernels to the bioassay dish, making sure to only place one kernel into each cap.
Add 30 ml of deionized water to the bottom of each bioassay tray. And incubate at 31 degrees Celsius for seven days. After seven days of inoculation with A.flavus, take pictures of four kernels that were designated for microscopic analysis and photography.
Then use a soft tissue and deionized water to clean the exterior of kernels. Perform longitudinal sections of kernels and immediately take photographs under the fluorescent microscope. To prepare the kernels for analysis, clean the outside of the remaining kernels with a soft tissue and deionized water.
Place four kernels, which constitute one rep, into a 15 ml screw cap polycarbonate vial containing two stainless steel balls. Immediately freeze the vials in liquid nitrogen and store at 80 degrees Celsius until ready to proceed with further processing and analysis. When ready, remove the kernels from the freezer and use a homogenizer to grind them at 1500 rpm for three minutes.
Use the same ground materials for GFP analysis, Aflatoxin determination, and molecular analysis, so that all values are representative of the samples. While a minimum of three replicates are recommended, more is always better depending on the availability of good quality kernels. In this study immature embryos of maize HI-II lines are transformed using Agrobacterium tumefaciens EHA101 strain containing the final plant destination vector expressing the Lablab purpureus AILP gene under the control of the 35S promoter from the cauliflower mosaic virus.
PCR amplification of the target AILP gene showed a 548 bp amplicon observed only in the transgenic maize lines. The AILP gene showed expression in the transgenic lines whereas no AILP expression was observed in the control plants. Early stage germinating spores are exposed to crude leaf extracts from young transgenic maize plants for 20 hours.
High full length from spores exposed to transgenic leaf extracts show a 58 to 80%reduction compared to the negative control. The A.flavus strain used contains a GFP reporter which enables the monitoring and quantification of fungal growth in real-time. Significant reduction in GFP fluorescence is observed in transgenic AILP kernels compared to the isogenic negative control.
A significant reduction is seen in fungal growth for AILP lines four and five, 69%and 72%respectively. The other lines show a reduction of 35 to 60%reduction in comparison to the control kernels. A reduction of 62 to 88%in Aflatoxin content was observed in the AILP kernels as compared to the control.
Line four is seen to have the lowest Aflatoxin content at 486 ng per g, followed by other lines ranging which had contents ranging between 640 and 1498 ng per g in the kernels. Following this procedure, additional methods can be performed such as using a fluorometer to quantify GFP and as its fungal growth. Immunobased commercial kits or HPLC or EPLC can be used to perform Aflatoxin quantitation.
The availability of KSA provides a quick and simple means of determining resistance to Aflatoxin contamination. The of different maize breeding lines can be assessed quickly using this technique. While the kernel screening assay is not hazardous, the operator should protect himself or herself from exposure to Aspergillus flavus spores, use proper protective equipment.
