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. 2022 Feb 7;12(3):62. doi: 10.1007/s13205-022-03128-z

Development and validation of rapid and cost-effective protocol for estimation of amylose and amylopectin in maize kernels

Shashidhar Bayappa Reddappa 1, Rashmi Chhabra 1, Zahirul Alam Talukder 1, Vignesh Muthusamy 1, Rajkumar Uttamrao Zunjare 1, Firoz Hossain 1,✉
PMCID: PMC8821753  PMID: 35186659

Abstract

Maize possesses wide variation in amylose and amylopectin which assumes significance as a part of both food-chain and different industrial applications. Estimation of amylose and amylopectin in maize kernels is important for developing suitable hybrids. The existing protocols for estimation of amylose and amylopectin in maize are elaborate and lengthy, and involve high cost. Here, we developed a rapid and cost-effective method for estimation of amylose and amylopectin in maize kernels. 10% toluene and 80% ethanol were used for removal of proteins (~ 10%) and lipids (~ 4%) from maize flour. The over-estimation of amylose was minimized using NaOH with KI to stop free KI to bind with amylopectin. Standards were improved by mixing amylose and amylopectin in different concentrations (0–100%), rather than using amylose or amylopectin alone. Standard curve generated regression equation of y = 90.436x + 0.8535 with R2 = 0.9989. Two types of samples viz., (1) protein, amylose and amylopectin (2) amylose and amylopectin, showed that starch fractions were highly comparable to expected values with correlation coefficient (r) of 0.9998 and mean standard deviation of 0.54. The protocol successfully estimated wide range of amylose (2.79–50.04%) and amylopectin (59.96–97.21%) among diverse maize inbreds including amylose extender1 (ae1) and waxy1 (wx1) mutants. Present protocol required 75% less time and 92.5% less cost compared to existing protocols. The newly developed method would be highly useful in developing maize hybrids high in amylose or amylopectin. This is the first report of rapid and cost-effective protocol for estimation of starch fractions in maize kernels.

Keywords: Amylopectin, Amylose, Kernels, Maize, Protocol, Starch

Introduction

Starch is a major energy reserve in higher plants and most abundant carbohydrates next to cellulose (Zhong et al. 2021). Starch is generally synthesized in a specialized plastid called amyloplasts with composition of amylose and amylopectin (Hossain et al. 2019). Amylose is a linear homopolymer of glucopyranose units linked by α-(1,4) linkage, whereas amylopectin is a branched homopolymer of glucopyranose with α-(1,4) and α-(1,6) linkage (Lin et al. 2019). The ratio of amylose and amylopectin plays an important role in determining the structure of starch granule. Maize starch is composed of an average of ~ 25% of amylose and ~ 75% of amylopectin (Hasjim and Jane 2009; Chung et al. 2009).

High amylose maize starch has wide application in making of candies, gums, adhesives and biodegradable plastics (Zhang et al. 2018). Amylose also contributes to Resistant Starch (RS) which takes > 120 min to digest in the human gut (Englyst et al. 1992). ‘Diabetes mellitus-type2’ has been identified as the most widespread lifestyle disease among humans affecting 366 million people worldwide. This figure is expected to rise to a level of 552 million by 2030 (Sami et al. 2017). Amylose content shows positive correlation with RS and total dietary fiber (TDF) (Xia et al. 2018). RS significantly lowers down the glycaemic index (GI) in humans (Behall and Hallfrisch 2002). The GI of traditional maize is 81, while the GI of high amylose maize reduces the GI to 44 (Ai and Jane 2016). Thus, high amylose maize is ideal food for the diabetic people. Apart from hypoglycaemic effect, high amylose maize also shows hypocholesterolemic effect by reducing cholesterol accumulation which further helps in reduction of bile-stone formations (Hashimoto et al. 2006; Malhotra 1968). On the other hand, maize with high amylopectin also called ‘waxy maize’ is a popular choice of food in South-East Asian countries (Xiaoyang et al. 2017). Waxy maize is consumed as ‘green corn’, especially during breakfast, and also popular as vegetable (Devi et al. 2017). Amylopectin possesses the property of high viscosity and is easily digested in human gut (Lu and Lu 2012). Amylopectin also serves as a popular ingredient in textile, adhesive and paper industries (Bao et al. 2012).

Amylose and amylopectin generally vary from 7 to 35% and 75 to 95%, respectively in maize (Li et al. 2018; Qi et al. 2020). However, Zhang et al (2020), while analyzing a set of maize inbreds reported amylose up to 67%, while waxy maize can have amylopectin up to 100% (Zhou et al. 2016). Precise estimation of amylose and amylopectin from the maize grain sample assumes great significance for the identification of suitable donor in the breeding program followed by development of high amylose or amylopectin maize. Various methods including colorimetric, potentiometric or amperometric titration techniques are available for determining of amylose and amylopectin (Bates et al. 1943; Williams et al. 1958). Colorimetric estimation is the widely and most frequently used technique, which is based on the ability of amylose to form complex with iodine to give blue color. However, it has been reported that even amylopectin can form complex with iodine and absorb minute quantity of light which interfere in amylose estimation (Davis et al. 1994). Amylose or amylopectin in most of the cereals is estimated either by extracting starch and then estimating or directly using the milled powder. In the first case, extraction of starch is tedious and requires lot of time, whereas in the second case due to the presence of proteins and lipids the estimated value of amylose is affected. Cereals like rice grains has nearly 90% of starch which makes needless to extract starch hence can be directly ground and used for estimation. However, in crop such as maize which is composed of 10% protein and 4% lipid with 70% of starch, it becomes necessary to remove protein and lipid prior to estimation of amylose or amylopectin. ‘Megazyme’ amylose kit based on the specific precipitation of amylopectin by concanavalin-A lectin has emerged as a popular choice in lab analysis (Yun and Matheson 1990; Gibson et al. 1997). However, the available protocols for estimation of amylose and amylopectin are time consuming and involves cumbersome methodology and costly chemicals (Zhu et al. 2008; Megazyme 2018). The present study was, therefore, undertaken to develop and validate a rapid, simple and cost-effective protocol that can be used for estimation of amylose and amylopectin in maize kernels.

Materials and methods

Preparation of standard curve

Amylose and amylopectin standards from maize with purity of > 99% from Sigma Life Sciences were used in preparation of standard curve. Standard curve was prepared by mixing relative proportion of amylose and amylopectin standards. For example, 60% amylose standard included 40% of amylopectin. Standard curve was plotted using amylose:amylopectin ratio of 100:0, 80:20, 60:40, 50:50, 40:60, 30:70, 25:75, 20:80, 10:100 and 0:100. Three replicates were used for accurate measurement of absorbance.

Preparation of standard sets for validation

Amylose and amylopectin standards from maize with purity of > 99% were used in preparation of starch standard mixtures. Bovine serum albumin protein (HiMedia Laboratories Pvt. Ltd.) with purity of > 98% was used. For testing the effectiveness of estimation method, two types of test samples were prepared with different contents of (1) protein (ranging from 5 to 15%), amylose and amylopectin (set 1–set 3), and (2) amylose and amylopectin (set 4–set 11) (Table 1). The analysis was carried out in four biological replicates.

Table 1.

Details of sets of standards used for validation

Sample Std. starch mixture (Amy + Amp + Protein) (%) Protein lost (%)a Expected amylose (%) Estimated amylose (%) SD Relative SD Amy (%)
Set-1 23.75 + 71.25 + 5.00 5.42 25 25.01  ± 0.70 2.79
Set-2 22.5 + 67.5 + 10.00 11.4 25 24.62  ± 0.51 2.09
Set-3 21.25 + 63.75 + 15.00 14.34 25 25.84  ± 0.55 2.14
Mean 25.15  ± 0.58 2.34
SE 0.23 – –
CD (5%) 0.54 – –
Set-4 90 + 10 + 0 – 90 92.44  ± 1.05 1.13
Set-5 75 + 25 + 0 – 75 76.56  ± 0.59 0.77
Set-6 60 + 40 + 0 – 60 60.44  ± 0.45 0.75
Set-7 50 + 50 + 0 – 50 50.51  ± 0.62 1.22
Set-8 40 + 60 + 0 – 40 40.51  ± 0.15 0.38
Set-9 30 + 70 + 0 – 30 30.18  ± 0.42 1.39
Set-10 20 + 80 + 0 – 20 21.00  ± 0.65 3.10
Set-11 10 + 90 + 0 – 10 10.16  ± 0.28 2.76
Mean 47.73  ± 0.52 1.44
SE 0.40 – –
CD (5%) 0.85 – –
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aProtein lost was calculated as the amount of weight lost after toluene treatment

Std standard, Amy amylose, Amp amylopectin, SD standard deviation, SE standard error, CD critical difference

Preparation of diverse maize samples for validation

Further, eight diverse maize inbreds of exotic and indigenous origin were used for validation. Of these, PMI-ae1-145, UMI-1200, BML-6, PMI-LAMY-1 and PMI-wx1-301 were developed by different breeding centers in India, while three lines (CML-580, CML-313 and CML-247) were developed by International Maize and Wheat Improvement Center (CIMMYT) Mexico. Among the inbreds, PMI-ae1-145 possessed mutant amylose extender1 (ae1) gene, while PMI-wx1-301 had the recessive waxy1 (wx1) allele. Rest of the inbreds possessed the wild-type Ae1 and Wx1 alleles. The experiment was undertaken with four biological replications. These inbreds with varying amylose and amylopectin were purposefully selected based on preliminary analysis in our laboratory.

Equipments and reagents

Seed miller with filter of diameter < 0.2 mm, vortex, centrifuge, heating water bath, spectrophotometer and tubes (2 ml and 50 ml) were required. Further, the protocol also required 80% ethanol (stored at either 4°C or room temperature more than a year), 0.1 M sodium chloride solution (0.1 M NaCl) containing 10% toluene (can be stored for one month in 4°C and care should be taken from direct sunlight exposure and kept away from heat), 1 M sodium hydroxide (1 M NaOH) (can be stored at room temperature for more than a month), 1 N acetic acid (1 N-AA) (can be stored at 4°C or room temperature and is extremely stable) and potassium iodide solution (KI, 0.26 g of Iodine in 10 ml of potassium iodide solution containing 2.6 g of KI) (should be freshly prepared before use and kept out of light). All the chemicals and reagents used in the protocol were purchased from HiMedia Laboratories Pvt. Ltd.

Amylose estimation protocol

Seed powder was obtained by milling 4–5 completely dried maize seeds having moisture content 10–12% with a diameter of < 0.2 mm. Approximately 100 mg of seed powder was transferred into 2 ml tubes. Sample was treated with 80% ethanol of 500 µl and vortexed for a short period of time (2 min). The sample tubes were centrifuged for 5 min at 10,000 rpm and supernatant was separated. It was repeated two times. The pellet obtained was further treated with 500 µl of 0.1 M NaCl solution containing 10% toluene and vortexed. The tubes were centrifuged for 5 min at 10,000 rpm and supernatant was discarded. It was repeated until the supernatant was clear of white milky layer. Obtained residue was again washed with 80% ethanol and dried completely in incubator at 80 °C for 4 h. The residue, thus, obtained is starch with < 5% impurity of fiber or thick outer covering of kernel.

Starch residues of 25 mg was carefully measured and transferred into 50 ml tubes. It was solubilized with 2.5 ml 1 M NaOH and was kept in hot boiling water bath for at least 15 min with intermittent shaking. Samples were removed from water bath, cooled at room temperature and volume was adjusted to 25 ml using double-distilled water. An aliquot of 1.25 ml was transferred to new 50 ml tube and treated with 150 µl 1 N acetic acid, 100 µl 1 M NaOH and 500 µl KI solution and made up to 25 ml using double-distilled water. The solution was incubated at room temperature for at least 20 min to develop color and absorbance was measured at 620 nm. Amylose was expressed as percentage by calculating the proportion of amylose over 25 mg starch residues taken in the final step. The procedure has been represented as a flow chart in Fig. 1.

Fig. 1.

Fig. 1

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Flow chart for the procedure for estimation of amylose

Statistical analysis

Mean, standard error (SE) and critical difference (CD) were analyzed using Windostat v8.0. Standard deviation (SD) between the replicated and relative standard deviation were calculated using MS-Office Excel-2019.

Estimation of amylopectin

Amylopectin was estimated by subtracting the value of amylose from the total 25 mg starch residues taken for final analysis.

Comparison with existing protocol

The newly developed protocol for the estimation of amylose in maize grains were also compared with (1) dual-wavelength method (Hovenkamp-Hermelink et al. 1988; Zhu et al. 2008; Zeng et al. 2012) and (2) GOPOD reagent and concanavalin-A-based Megazyme kit (© Megazyme 2018) for its efficacy besides time and cost analysis.

Results

Preparation of standard curve

Standard curve with different concentrations of standard amylose and amylopectin in triplicates (100:0, 80:20, 60:40, 50:50, 40:60, 30:70, 25:75, 20:80, 10:100 and 0:100) was prepared, which generated regression equation of y = 90.436x + 0.8535 with R2 value of 0.9989 (Fig. 2).

Fig. 2.

Fig. 2

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Standard curve for estimation of amylose

Validation of developed protocol

Estimation of amylose and amylopectin in different sets of standard samples

Two types of test samples were prepared with different mixtures of (1) protein, amylose and amylopectin (set 1–set 3) (2) amylose and amylopectin (set 4–set 11) for validating the protocol. All the eleven sets showed amylose and amylopectin concentrations comparable to expected values with correlation coefficient of 0.9998 and mean standard deviation was 0.54. Repeated analysis for the standard starch samples yielded repeatability with relative standard deviation of 1.68% (Table 1).

Estimation of amylose and amylopectin in diverse maize samples

Eight diverse maize genotypes were employed for estimation of amylose and amylopectin in maize kernels. The amylose ranged from 2.79% in waxy line (PMI-wx1-3) to 50.04% (PMI-ae1-145) with mean standard deviation of 0.62. While, amylopectin varied from 49.96% (PMI-ae1-145) to 97.21% (PMI-wx1-301). Other genotypes with different proportion of amylose and amylopectin included CML-580 (amylose: 35.59%, amylopectin: 64.41%), CML-313 (amylose: 29.77%, amylopectin: 70.23%), UMI-1200 (amylose: 24.18%, amylopectin: 75.82%), BML-6 (amylose: 21.69%, amylopectin: 78.31%), CML-247 (amylose: 19.74%, amylopectin: 8026%) and PMI-LAMY-1 (amylose: 12.13%, amylopectin: 87.87%). Repeated analysis of these maize samples yielded repeatability with relative standard deviation of 4.08% (Table 2).

Table 2.

Estimation of amylose and amylopectin in diverse maize inbreds

Inbreds Amylose (%) Amylopectin (%) SD Amylose (%) Amylopectin (%) SD Amylose (%) Amylopectin (%) SD
Present study Dual wavelength Megazyme
PMI-ae1-145 50.04 49.96  ± 0.28 47.93 52.07  ± 0.60 49.96 50.04  ± 1.01
CML-580 35.59 64.41  ± 1.08 33.19 66.81  ± 0.46 35.46 64.54  ± 0.43
CML-313 29.77 70.23  ± 0.62 28.23 71.77  ± 0.55 29.55 70.45  ± 0.74
UMI-1200 24.18 75.82  ± 0.53 22.18 77.82  ± 0.67 24.85 75.15  ± 0.72
BML-6 21.69 78.31  ± 0.59 20.57 79.43  ± 0.83 22.44 77.56  ± 0.60
CML-247 19.74 80.26  ± 1.00 18.54 81.46  ± 0.49 19.68 80.32  ± 0.74
PMI-LAMY-1 12.13 87.87  ± 0.47 11.65 88.35  ± 0.77 12.55 87.45  ± 0.49
PMI-wx1-301 2.79 97.21  ± 0.36 2.15 97.85  ± 0.19 2.79 97.21  ± 0.25
Mean 24.49 75.50  ± 0.62 23.05 76.94  ± 0.58 24.66 75.34  ± 0.62
SE 0.50 0.50 0.50 0.50 0.49 0.49
CD (5%) 4.74 4.74 4.74 4.74 4.74 4.74
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SD standard deviation, SE standard error, CD critical difference

Comparison with existing protocols for amylose and amylopectin

The time taken for completion of this developed protocol for estimation of amylose and amylopectin starting with the defattening of flour up to final estimation was approximately 8 h for 100 samples, whereas the Megazyme’s kit took much longer time up to 24 h for 24 samples (© Magazyme 2018). The protocol reported here required a cost of US$ 0.25 per sample compared to US$ 3.20 per sample using Megazyme’s kit (© Megazyme 2018) (Table 3). On the other hand, dual-wavelength method involved a cost of US$ 0.49 per sample, with a requirement of 4.3 h for 100 samples. Interestingly, it was found that values of the amylose estimated in the protocol developed here were similar to the values measured through Megazyme’s kit, while the values of amylose using dual-wavelength method were quite lower than both protocols.

Table 3.

Cost and time analysis of the developed protocol

Cost analysis
Present study Dual wavelength Megazyme
Chemicals Cost in INR (100 samples) Cost in INR (100 samples) Cost in INR (24 samples)
Ethanol 1155.00 2310.00 -
Sodium chloride 0.75 – -
Toluene 21.45 – -
Sodium hydroxide 6.00 12.00 -
Glacial acetic acid 14.11 28.20 -
Potassium iodide 362.15 724.30 -
Iodine 27.00 54.00 -
Amylopectin standard 24.10 48.20 -
Amylose standard 250.00 500.00 -
Total cost

1860.56

(INR 18.60 per sample)

US$ 25.36

(US$ 0.25 per sample)

3676.70

(INR 36.77 per sample)

US$ 48.99

(US$ 0.49 per sample)

5760.00

(INR 240.00 per sample)

US$ 76.74

(US$ 3.20 per sample)
Time analysis
Preparation of reagents 0.3 h 0.3 h 2 h
Procedure 7.3 h 4.0 h 22 h
Total time 7.6 h (~ 8 h) 4.3 h 24 h
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1 US$ = INR 75.05

Discussion

Amylose possesses linear helical configuration which makes a stable compact structure resistant to digestive enzymes, thereby lowering the GI (Wee and Henry 2020). The GI of traditional maize is 81, while the GI of high amylose maize is reduced to 44 (Ai and Jane 2016). Similarly, white rice and brown rice have a GI of 78 and 65, respectively. Whereas, high amylose rice possesses low GI of 39 (Rohman et al. 2014; Wee and Henry 2020). The present investigation was targeted to develop a rapid, simple and cost-effective protocol for estimating amylose and amylopectin in maize kernels. Different methods were tested with standards to get reproducible and robust results, viz., Iodine-DMSO colorimetric method in combination with calcium chloride (Knutson and Grove, 1994), potassium iodide method (Jain et al. 2012) and Megazyme kit for estimation of amylose and amylopectin (© Megazyme 2018). Moreover, various reports were carefully studied for different steps starting from defatting and deproteinization of maize flour. Zhu et al. (2008) followed aqueous leaching process given by Mua and Jackson (1998) to get purified amylose fractions. Jane et al. (1992) employed general procedure given by Schoch (1942) to fractionate starch of normal corn, potato and normal rice which takes approximately 40 h for fractionation into amylose and amylopectin. Since protein is present in maize kernels in significant amount (~ 10%), deproteinization of maize flour is an essentiality.

Baker et al. (1979) used various alcohols and solvents in different concentrations in order to remove oil from grain sample. Here, we optimized 80% ethanol for defattening of maize flour. While, deproteinization of maize flour has been standardized with 0.1 M NaCl with 10% toluene. This step of removal of proteins has not been reported in many of the amylose estimating protocols. Toluene is more effective with the use of NaCl, as the solubility of proteins is increased by using low concentration (0.1 M NaCl) of salts which increases the interaction between the protein and the solvent (salting-in). On the other hand, toluene being an organic solvent denatures protein and forms a milky layer which is ultimately discarded. Repetition of this step aids in removal of protein. As organic solvents denature proteins by disrupting hydrophobic interaction between non-polar side chains of proteins, toluene was also suggested to be a potential solvent for protein denaturation (Asakura et al. 1978). This step was optimized and repeated thrice in case of maize flour until clear toluene phase was obtained. The clarity of the toluene phase depends upon the content of protein, therefore, the repetitions of treatment with 0.1 M NaCl with 10% toluene would vary depending upon the protein content in other cereals.

After the removal of fats and proteins from the maize flour, amylose was estimated using spectroscopic method. Amylopectin forms complex with iodine which shows absorption at 620 nm thereby interfering with the precise estimation of amylose (Gibson et al. 1997; Jain et al. 2012). In earlier reports, no step was involved to avoid the formation of this complex. In our study, we suggested to add NaOH along with KI, which initiates a reaction with iodine complexed with amylopectin, resulting in a clear solution with no absorption at 620 nm. The remaining NaOH in the solution was neutralized by acetic acid.

Preciseness of the developed protocol

For accuracy and precision, preparation of standards was also improvised by mixing amylose and amylopectin in different concentrations, rather than using amylose alone. By employing this strategy, interference due to absorbance by amylopectin was further minimized to a greater extent. In terms of accuracy, as mentioned in Megazyme’s kit protocol, repeated analyses of a set of samples yielded repeatability (within laboratory) with relative standard deviations of < 5% for pure starch and ~ 10% for cereal flours (© Magazyme 2018). In our protocol, the repeated analysis for the standard starch samples yielded repeatability with relative standard deviation of 1.68% and it was 4.08% for maize samples. This depicts the robustness and reproducible nature of the newly developed protocol.

Variation in amylose and amylopectin in maize genotypes

Wide variation was observed among the eight genotypes. These inbreds were deliberately selected based on our preliminary analysis among a set of ~ 300 lines available with us. Among the lines, PMI-wx1-301 possessed the recessive wx1 gene which conferred highest amylopectin. The gene Wx1 located on chromosome-9 encodes a granule-bound starch synthase (GBSS-I), which catalyses amylose synthesis from ADP-glucose in the endosperm (Mason-Gamer et al. 1998). The recessive wx1 allele inhibits the conversion thereby leading to the increased amylopectin (Hossain et al. 2019). On the other hand, the recessive ae1 gene present in chromosome-5 codes for starch branching enzyme (sbeIIb) that enhances amylose to a level of > 50% (Li et al. 2008). Other inbreds possessed amylose in the range of 10–20%, 20–30% and 30–40%, while amylopectin varied from 60 to 70%, 70 to 80% and 80 to 90%. Wide variation of amylose and amylopectin has been reported in diverse maize germplasm (Li et al. 2018). This suggests that wide variability of amylose and amylopectin in maize genotypes including the recessive wx1 and ae1 genes can be effectively measured using our newly developed protocol.

Cost and time analysis

The analysis showed that the procedure developed here takes ~ fourfold less time and ~ 13-fold less cost as compared to Megazyme kit. On the other hand, though dual-wavelength method required nearly half of the time compared to the newly developed method, its cost is double the cost of the new protocol. Further, the amylose data generated in the newly developed method suggested that values were at par with the Megazyme kit and, thus, was highly reliable. On the other hand, amylose content measured through dual-wavelength method was quite low as compared to the Megazyme kit as well as the present protocol. The lower values of amylose obtained in dual-wavelength method are possibly due to non-separation of starch and protein from flour sample during the process. In our method developed here, we included steps to remove proteins and separated starch from the flour sample which gave the amylose values highly comparable to widely used Megazyme kit. In any breeding program, large number of genotypes is required to be screened for amylose and amylopectin in a shorter time with less cost. The present protocol is simple as it requires very basic and less costly equipments. Further, the method is rapid and cost-effective, besides being robust and reproducible. These features of the protocol suite the need of any breeding program to select for the promising inbreds followed by development of amylose- or amylopectin-rich maize. This rapid and cost-effective protocol can also be used in estimation of amylose in other cereals such as rice which possess similar to maize in grain composition with ~ 70–75% starch, 7–10% protein and 2–3% oil (Chiang et al. 2005). Due to these similar grain composition, Megazyme’s kit is universally used for estimation of amylose in rice as well (© Megazyme 2018). In rice as well, increase in amylose proportion leads to lower GI (Wee and Henry 2020), while lowering of the same leads to stickiness while cooking (Zhang et al. 2021). Thus, analysis of diversity of amylose in rice accession using this newly developed protocol assumes great significance in breeding program.

Conclusion

The present study reported the development of protocol for rapid estimation of amylose and amylopectin in a time- and cost-effective manner. The protocol has been validated with the mixtures containing different amylose and amylopectin content and diverse set of maize inbreds with varying degree of starch fractions. The results were robust and reproducible with standard deviation of 1.68% in standards and ~ 4.08% in unknown maize flour samples. It required 49–92% less cost compared to the existing methods. The developed method would be useful in maize breeding programs targeting on high amylose and amylopectin. This is the first report of development and validation of protocol for rapid and cost-effective method for estimation of amylose and amylopectin in maize kernels.

Acknowledgements

First author thanks Indian Council of Agricultural Research (ICAR), New Delhi for providing the fellowship during the M.Sc. program. Financial help received from Division of Genetics, ICAR-IARI, New Delhi, and ICAR-Consortia Research Platform on ‘Molecular breeding for improvement of tolerance to biotic and abiotic stresses, yield and quality traits in crops - Maize component’ (IARI Project Code No.: 12-143C) is thankfully acknowledged. We thank breeders of the national program and CIMMYT, Mexico for sharing the inbreds.

Author contributions

Conduct of experiment: SBR; development of protocol: RC, SBR; validation of protocol with maize inbreds: ZAT; development and maintenance of inbreds: VM; Statistical analysis: RUZ; Manuscript writing: SBR, RC and FH; Design of experiment: FH.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Contributor Information

Shashidhar Bayappa Reddappa, Email: shashintrsachin@gmail.com.

Rashmi Chhabra, Email: reshu0428@rediffmail.com.

Zahirul Alam Talukder, Email: zahirbari@yahoo.com.

Vignesh Muthusamy, Email: pmvignesh@yahoo.co.in, Email: vignesh@iari.res.in.

Rajkumar Uttamrao Zunjare, Email: raj_gpb@yahoo.com.

Firoz Hossain, Email: fh_gpb@yahoo.com.

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