Abstract
Our goal was to improve sweet corn (Zea mays L.) by the novel approach of combining three grain mutants in one plant, shrunken?2 (sh2sh2), purple (Pr1Pr1C1C1), and opaque?2 (o2o2), for increased sugar, anthocyanin, and tryptophan content, respectively. We crossed purple opaque?2 waxy maize with sweet corn inbred lines. In the segregating selfed progenies, the opaque?2 gene was detected by the genetic marker phi057, the two other genes by the visual markers purple and shrunken. The purple opaque?2 sweet corn performed well in field tests; it had high total sugar content as required for sweet corn, improved protein quality indicated by 30% higher tryptophan content, and a 10?fold higher anthocyanin content than yellow kernel maize. This new sweet corn germplasm is a first step toward developing specialty maize with increased health benefits.
Abbreviations
DAP
days after pollination
QPM
quality protein maize
1 BACKGROUND
Sweet corn (Zea mays L.) is an important source of fiber, minerals, and certain vitamins (Lertrat & Pulam, 2007). In the United States, the fresh market accounts for nearly 70% of the total production of sweet corn, the United States’ second?largest processed vegetable crop (Hansen, 2019). The shrunken?2 homozygous recessive gene promotes a high level of sucrose. It retains higher sugar and moisture content for longer than other mutants such as bt and su1 (Creech, 1965). In Thailand, sweet corn with the shrunken?2 gene accounts for about 95% of total consumption. In southeast Asia, it competes with the traditional waxy maize, which is used as both a staple food and a snack (Sinkangam et al., 2011). Anthocyanins are among the most important pigments of vascular plants (Castaneda?Ovando et al., 2009), contributing to high antioxidant levels that can reduce cancer cell proliferation and inhibit tumor formation (Cevallos?Casals & Cisneros?Zevallos, 2003; Li, Walker, & Faubion, 2011). Purple maize has high concentrations of anthocyanins that accumulate in the aleurone layer and kernel pericarp (Li et al., 2011). Three genes, Pr1, C1, and R1, affect the production of anthocyanin; there is no anthocyanin in the kernel pericarp when homozygous recessive alleles of r1 or c1 are present (Rosemary, 2000). Anthocyanin biosynthesis in maize is transcriptionally regulated by an interaction between two sets of transcription factors (Chandler, Radicella, Robbins, Chen, & Turks, 1989). The yellow or white colors of the starchy endosperm are seen when the aleurone layer is colorless. In comparison to other cereals, the quantity of grain protein is low in common maize (8–9%). Its quality is poor as well due to low lysine and tryptophan content (FAO, 1992). Quality protein maize (QPM) is homozygous for the recessive opaque?2 gene (Nelson, Mertz, & Bates, 1965). This common name was introduced to mark the high increase in the nutritional value of the grain protein. A codominant marker, phi 057, can detect homozygous dominant (O2O2), heterozygous (O2o2), and homozygous recessive (o2o2) plants separately (Ribaut & Hoisington, 1998). The protein content is less affected by QPM; however, both lysine and tryptophan are significantly increased (Nelson et al., 1965). The combination of all three grain quality mutants described above, and their successful expression, has not been shown before. It was our objective to prove the feasibility of a purple opaque?2 sweet corn (Pr1Pr1, C1C1, o2o2 and sh2sh2) with improved health benefits by pyramiding these genes.
2 MATERIAL AND METHODS
2.1 Plant material and cultivation
From the breeding program of the Kasetsart University, purple opaque?2 waxy maize (Pr1Pr1, C1C1, o2o2 and wxwx) was crossed with sweet corn inbred lines to obtain F1 hybrids. Five generations of selected plants were self?pollinated to obtain the S5 lines having purple and red kernels combined with opaque?2 and shrunken?2 genes. A commercial sweet corn hybrid with yellow kernels was used as a check. The field experiments were conducted at the Research Center of Kasetsart University, Thailand, between 2012 and 2015. Two experimental yield trails were arranged in a randomized complete block design with four replications in September 2014 and February 2015. Each plot consisted of four rows, 5 m long and 0.75 m apart, with 21 plants per row. Ten ears from two middle rows of each plot were harvested as the samples for chemical analysis. The cultivation conditions were identical to those described for the combination of waxy starch quality and QPM (Sinkangam et al., 2011). The one difference was the earlier harvest date for sweet corn, 3 wk after flowering.
2.2 SSR marker assay for the opaque?2 locus
The opaque?2 allele was detected by the SSR marker phi057 from young and healthy leaves of individual 4?wk?old plants, as described in detail by Sinkangam et al. (2011). The amplified fragment was about 142–157 bp. The amplification was performed in 20 μl containing 3 mM MgCl2, 1 U Taq DNA polymerase, 0.2 mM dNTPs, 10 pM of each primer, and 50 ng of template DNA and distilled water.
2.3 Analysis of total sugars and reducing sugar
Kernels were harvested 20 d after pollination (DAP). Samples of 20–25 grams of seeds were chopped, crushed, and percolated through a cotton membrane. The suspension was poured into tubes and centrifuged at 10,000 rpm at 4 °C for 20 min. Further analysis was carried out, according to Nelson (1979).
Core Ideas
We tested the feasibility of combining three grain mutants in sweet corn.
The new sweet corn germplasm has improved antioxidant levels and protein content.
Tests indicate the agronomic vigor of this new germplasm.
2.3.1 Total sugars
One milliliter of solution was placed in a tube, and 0.5 ml 0.1 M HCl was added; the solution was boiled at 100 °C for 15 min. After cooling, the solution was treated and measured spectrophotometrically at 500 nm (Nelson, 1980).
2.3.2 Reducing sugar
Reducing sugar analysis was done, according to Nelson (1980). One milliliter of alkalic copper reagent was added to the solution, boiled at 100 °C for 15 min, and cooled at room temperature. Finally, a sample was measured by the spectrophotometer at 500 nm; the resulting absorbance value was compared with the standard D?glucose solution as milligrams glucose per milliliter.
2.4 Analysis of tryptophan and total protein
The pericarp and embryo of the random samples of 40 kernels per line were removed. The remaining kernels were soaked in distilled water for 25 min before removing pericarps and embryos. The ground samples were then analyzed for tryptophan content, as described by Nurit, Tiessen, Pixley, and Palacios?Rojas (2009); the protein content was analyzed using the micro?Kjeldahl method (Bailey, 1967).
2.5 Analysis of anthocyanin content
A sample of 30 kernels per line was dried at 40 °C for 3 d. The kernels were ground in a cyclone mill. Each sample was weighed out to 0.02 g of powder in an Eppendorf tube, and 1.3 ml of 1% (v/v) trifluoroacetic acid (TFA) was added. Then, after being vortexed, the samples were placed horizontally on ice at 4 °C and shaken for 90 min at 150 rpm. After the ice incubation, the tubes were centrifuged at 14,000 rpm for 5 min. The supernatant was measured by a spectrophotometer at 520 nm. The result of the absorbance value was compared with the standard pelargonidin (Galicia, Nurit, Rosales, & Palacios?Rojas, 2009).
2.6 Statistical analysis
Data were analyzed according to the combined analysis of randomized complete block design using R statistics (R Core Team, 2019).
3 RESULTS
The combination of three different quality traits is expressed by different combinations of dominant and recessive alleles. Further studies demand homozygosity. In the S5 (fifth selfing generation), 226 plants were generated. From these, nine lines were selected that were homozygous in anthocyanin, six red ones (pr1pr1, C1C1, o2o2, and sh2sh2) and three purple ones (Pr1Pr1, C1C1, o2o2, and sh2sh2). No further segregation occurred. The complete breeding scheme is shown in Figure 1.
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FIGURE 1
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Selection scheme for purple opaque?2 sweet corn
Field vigor has not been assessed in detail as the experimental inbred lines cannot be compared with commercial hybrids. However, eight experimental hybrids were generated by crosses between them and tested under the same field conditions (data not shown). Their yield level (fresh ear with husk) was, on average, 10.4 t ha−1. The conventional hybrid yielded slightly more (11.5 t ha−1), indicating that the plant vigor of the experimental hybrids was still high. The purple and red opaque?2 sweet corn kernels of S5 lines were analyzed for total sugars, non?reducing sugars, anthocyanin, and the percentage of tryptophan in protein at 20 DAP (Table 1). This harvest date was same as that used in the preliminary tests. The total sugar content dropped by 36% from 20 to 29 DAP (data not shown). Except for the protein content, all grain kernel traits exhibited significant differences. The total sugar content was on average slightly lower in red and purple lines than in the commercial hybrid Insee 2, ranging from 207 to 464 mg g−1 in single genotypes. Values for non?reducing sugars are not shown as they correlated closely with total sugars (r = .95; P < .05). The anthocyanin content of red and purple kernels was least 25 times higher than in the yellow kernel genotype. The tryptophan content was significantly higher in the opaque?2 group, i.e., the red kernel lines, than in the non?opaque?2 commercial hybrid Insee 2.
TABLE 1. Kernel chemical composition for S5 lines of opaque?2 sweet corn with different kernel colors. The averages, minimum, and maximum values for red and purple kernel lines are shown
Total sugars Anthocyanin Tryptophan in protein Protein
Lines mg g−1 mg 100 g−1 % % dry wt. Kernel color
R (6 lines) 328 345 0.96 13.5 Red
R, max. 464 416 1.01 16.3 Red
R, min. 223 264 0.89 11.9 Red
P (3 lines) 275 417 0.95 12.9 Purple
P, max. 311 429 0.94 12.8 Purple
P, min. 207 402 0.92 11.3 Purple
Y (Insee 2) 358 15 0.72 16.1 Yellow
F?test ** ** * ns†
LSD(.05) 36.17 64.41 0.09 –
LSD(.01) 50.74 90.3 – –
CV (%) 5.26 10.42 4.14 6.51
Note. R, red kernels; P, purple kernels; Y, yellow kernels; –, not determined.
*, ** Significant at the .05 and .01 levels, respectively.
†ns, nonsignificant at the .05 level.
4 DISCUSSION
Our challenge was to increase the kernel quality of protein and the content of anthocyanin, requiring the successful combination of three mutant alleles using modern breeding technology. The opaque?2 gene was fixed in the S1 segregating population by using the marker phi057. This occurred in the early generation of self?pollination, proving the effectiveness of marker?assisted selection for detecting homozygous recessive plants by the phi057, as was done previously for opaque?2 waxy maize (Sinkangam et al., 2011) and field maize (Bantte & Prasanna, 2003; Jompuk, Cheuchart, Jompuk, & Apisitwanich, 2011). As the shrunken?2 (sh2sh2) gene was alone responsible for the sweet corn identity in our germplasm, the visual marker “wrinkled kernels” could be successfully used to select this allele in the S2 generation of dry ears. Shrunken?2 gives sweet corn two to three times more sucrose at harvest maturity (Creech, 1965). Our S5 lines of opaque?2 sweet corn varied greatly in total sugars and non?reducing sugar. However, their sweetness was in the normal range, providing sufficient genetic variability for further improvement. But allelic variation at other loci can profoundly influence sucrose and total sugar levels in freshly harvested sweet corn (Wong, Juvik, Breeden, & Swiader, 1994). For comparisons, the date of harvest is important; in our trials, the total sugar content dropped considerably after 20 DAP. The pr1 aleurone accumulated red pelargonidin glycosides, while Pr1 showed the presence of purple cyanidin glycosides (unpublished data). The anthocyanin content of red and purple kernels attained the intended high levels in both color groups, with more constant and very high values in purple kernels; the anthocyanin content was very low in yellow kernels. In field maize (Jompuk et al., 2011; Prasanna, Vasal, Kassahun, & Singh, 2001) and waxy maize (Sinkangam et al., 2011), QPM genotypes had almost double the amount of tryptophan compared with normal maize but were similar in overall protein content. In these cases, the kernels fully mature with well?developed storage glutens. In our opaque?2 sweet corn lines, increases in tryptophan were significant although less pronounced compared with the commercial hybrid (Insee 2, non?opaque?2). Nevertheless, the increase in protein quality was still satisfactory. In conclusion, we achieved our goal of a triple grain mutant. The development of a new model, sweet corn germplasm, purple opaque?2 sweet corn, with improved protein quality and antioxidant content, is a first step toward developing competitive hybrids with improved health benefits.
ACKNOWLEDGMENTS
This research was financially supported by the National Research Council of Thailand for the project research on “Sweet Corn Improvement for Increasing Anthocyanin and Tryptophan Contents in Kernel.”