**2. Discovery of aflatoxin-resistance**

#### **2.1. Traditional screening techniques**

Screening maize for resistance to kernel infection by *Aspergillus flavus* or for resistance to aflatoxin production is a more difficult task than most disease screening. Successful screen‐ ing in the past had been hindered [15] by the lack of 1) a resistant control; 2) inoculation methods that yield infection/aflatoxin levels high enough to differentiate among genotypes (natural infection is undependable); 3) repeatability across different locations and years; and, 4) rapid and inexpensive methods for assessment of fungal infection and aflatoxin lev‐ els. Several inoculation methods, including the pinbar inoculation technique (for inoculating kernels through husks), the silk inoculation technique, and infesting corn ears with insect larvae infected with *A. flavus* conidia have been tried with varying degrees of success [9, 16]. These methods can each be useful, however, clarity must exist as to the actual resistance trait to be measured (e.g. husk tightness; silk traits; the kernel pericarp barrier; wounded kernel resistance), before an appropriate technique can be employed. Silk inoculation, how‐ ever, (possibly more dependent upon the plant's physiological stage and/or environmental conditions) has proven to be the most inconsistent of the inoculation methods [17].

Plating kernels to determine the frequency of kernel infection and examining kernels for emission of a bright greenish-yellow fluorescence (BGYF) are methods that have been used for assessing *A. flavus* infection [15]. While both methods can indicate the presence of *A. fla‐ vus* in seed, neither can provide the kind of accurate quantitative or tissue-localization data useful for effective resistance breeding. Several protocols have been developed and used for separation and relatively accurate quantification of aflatoxins [18].

### **2.2. Early identification of resistant maize lines**

been identified through field screening, there is always a need to continually identify and

An important contribution to the identification/investigation of kernel aflatoxin-resistance has been the development of a rapid laboratory screening assay. The kernel screening assay (KSA), was developed and used to study resistance to aflatoxin production in GT*-*MAS:gk kernels [13, 14]. The KSA is designed to address the fact that aflatoxin buildup occurs in ma‐ ture and not developing kernels. Although, other agronomic factors (e.g. husk tightness) are known to affect genetic resistance to aflatoxin accumulation in the field, the KSA measures seed*-*based genetic resistance. The seed, of course, is the primary target of aflatoxigenic fun‐ gi, and is the edible portion of the crop. Therefore, seed*-*based resistance represents the core objective of maize host resistance. Towards this aim, the KSA has demonstrated proficiency in separating susceptible from resistant seed [13, 14]. This assay has several advantages, as compared to traditional field screening techniques [14]: 1) it can be performed and repeated several times throughout the year and outside of the growing season; 2) it requires few ker‐ nels; 3) it can detect/identify different kernel resistance mechanisms; 4) it can dispute or con‐ firm field evaluations (identify escapes); and 5) correlations between laboratory findings and inoculations in the field have been demonstrated. The KSA can, therefore, be a valuable complement to standard breeding practices for preliminary evaluation of germplasm. How‐

Screening maize for resistance to kernel infection by *Aspergillus flavus* or for resistance to aflatoxin production is a more difficult task than most disease screening. Successful screen‐ ing in the past had been hindered [15] by the lack of 1) a resistant control; 2) inoculation methods that yield infection/aflatoxin levels high enough to differentiate among genotypes (natural infection is undependable); 3) repeatability across different locations and years; and, 4) rapid and inexpensive methods for assessment of fungal infection and aflatoxin lev‐ els. Several inoculation methods, including the pinbar inoculation technique (for inoculating kernels through husks), the silk inoculation technique, and infesting corn ears with insect larvae infected with *A. flavus* conidia have been tried with varying degrees of success [9, 16]. These methods can each be useful, however, clarity must exist as to the actual resistance trait to be measured (e.g. husk tightness; silk traits; the kernel pericarp barrier; wounded kernel resistance), before an appropriate technique can be employed. Silk inoculation, how‐ ever, (possibly more dependent upon the plant's physiological stage and/or environmental

conditions) has proven to be the most inconsistent of the inoculation methods [17].

Plating kernels to determine the frequency of kernel infection and examining kernels for emission of a bright greenish-yellow fluorescence (BGYF) are methods that have been used for assessing *A. flavus* infection [15]. While both methods can indicate the presence of *A. fla‐ vus* in seed, neither can provide the kind of accurate quantitative or tissue-localization data

utilize additional sources of maize genotypes with aflatoxin-resistance.

ever, field trials are necessary for the final confirmation of resistance.

**2. Discovery of aflatoxin-resistance**

**2.1. Traditional screening techniques**

4 Aflatoxins - Recent Advances and Future Prospects

Two resistant inbreds (Mp420 and Mp313E) were discovered and tested in field trials at dif‐ ferent locations and released as sources of resistant germplasm [11, 19]. The pinbar inocula‐ tion technique was one of the methods employed in the initial trials, and contributed towards the separation of resistant from susceptible lines [11]. Several other inbreds, demon‐ strating resistance to aflatoxin contamination in Illinois field trials (employing a modified pinbar technique) also were discovered [12]. Another source of resistance discovered was the maize breeding population, GT-MAS:gk. This population was derived from visibly clas‐ sified segregating kernels, obtained from a single fungus-infected hybrid ear [10]. It tested resistant in trials conducted over a five year period, where a kernel knife inoculation techni‐ que was employed.

These discoveries of resistant germplasm may have been facilitated by the use of inocula‐ tion techniques capable of repeatedly providing high infection/aflatoxin levels for geno‐ type separation to occur. While these maize lines do not generally possess commercially acceptable agronomic traits, they may be invaluable sources of resistance genes, and as such, provide a basis for the rapid development of host resistance strategies to eliminate aflatoxin contamination.
