extfieldsofstudy: Chemistry Medicine

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Identification of key odorants in honeysuckle by headspace-solid phase microextraction and solvent-assisted flavour evaporation with gas chromatography-mass spectrometry and gas chromatograph-olfactometry in combination with chemometrics

At present, the identification of honeysuckle aroma depends on experienced tasters, which results in inconsistencies due to human error. The key odorants have the potential to distinguish the different species and evaluate the quality of honeysuckle. Hence, in this study, a more scientific approach was applied to distinguish various honeysuckles. The volatile compounds of different species and parts of honeysuckle were separately extracted by headspace-solid phase microextraction (HS-SPME) and solvent assisted flavor evaporation (SAFE). Compounds with greater volatility such as aldehydes, limonene, γ-terpinene, and terpinolene were preferentially extracted by HS-SPME. As a complementary extraction method to HS-SPME, SAFE was found to recover comparatively more polar compounds such as eugenol, decanoic acid, and vanillin. Subsequently, key odorants with the highest flavour dilution (FD) factors were detected by aroma extract dilution analysis (AEDA). These were benzaldehyde, 4-ethylphenol, decanoic acid, vanillin, 3-methyl-2-butenal, and β-ionone in honeysuckle flowers and γ-octalactone, 4-ethyl phenol, and vanillin in honeysuckle stem. Finally, principal component analysis (PCA) was conducted to analyze not only the key odorants of species and parts of honeysuckle but also their different origins. The results of PCA suggested that the species of honeysuckle contributed much more to variations in aroma rather than their origins. In conclusion, the application of the key odorants combined with PCA was demonstrated as a valid approach to differentiate species, origins, and parts of honeysuckle.

Introduction
Honeysuckle is a kind of herbal plant with many species and widely used and grown in northern hemisphere countries [1]. It has been consumed as traditional medicine and tea beverage for thousands of years [2]. In addition to the treatment of pneumonia and respiratory infections, and the improvement of consumers' health, honeysuckle possesses anti-inflammatory, antioxidant, and antiviral properties [3,4]. For this reason, it has been gradually added into foods and beverages to improve product value and to provide adjuvant therapy [5,6]. Recently, the aroma of herbal plants in food products has also increasingly gained popularity and consumer acceptance around the world [7]. Key aroma profiles helps to distinguish the different species and determines the quality of honeysuckle. At present, the quality of honeysuckle is evaluated by professional tasters that causes inconsistent and inaccurate results [8]. Therefore, a systematic study of the volatile compositions of different species, parts and origins of honeysuckle was conducted in this study.
The selection of an appropriate extraction method is a prerequisite to study volatile compounds in honeysuckle. The complex plant matrix requires the development of various sample preparation approaches to address the issue of sample complexity [9]. Some solvent extraction methods operate under high temperatures and use toxic organic solvents that are prone to thermal reactions and cause adverse effects to environment. In comparison, headspace-solid phase microextraction (HS-SPME) is a relatively low-temperature, solvent-free, and rapid approach [10]. For this reason, it is a popular method applied in the food matrix of natural materials [11,12]. HS-SPME is suitable for the isolation of highly volatile compounds. In comparison to HS-SPME, solvent-assisted flavour evaporation (SAFE) is able to extract less volatile and a greater range of polar compounds from complex matrices [13]. By applying high vacuum and low-temperature conditions, SAFE avoids aroma modification during the extraction of heat-liable aroma compounds from natural materials [14]. Besides, SAFE reduces the number of side reactions that may occur during aroma isolation, such as enzymatic degradation [15]. Hence, these two extraction methods are suitable for isolating volatile compounds from natural materials.
The potency of the aroma is often not proportionate to its peak area detected by chromatography-mass spectrometry (GC-MS) because many high abundance compounds may have a high odor threshold and thus do not serve as key odorants in the overall matrix [16]. Hence, it was difficult to distinguish between the aromas of different honeysuckle species using the volatile characteristics alone. As well known, aroma extract dilution analysis (AEDA) has been widely used to fill this gap by suggesting the contribution of each compound through the flavour dilution (FD)-factor [17]. The key odorants detected by AEDA may then be used to better describe the characteristic aroma of honeysuckle, and has the added potential to distinguish different species of honeysuckle. Therefore, the objective of this study was to extract the volatile compounds in various honeysuckles using HS-SPME in conjunction with SAFE, both coupled to GC-MS/FID. Furthermore, their key odorants were identified using AEDA. Lastly, principal component analysis (PCA) was performed to show the differences in the aromatic profiles of honeysuckle samples from different species, origins, and parts of the plants.

Sample preparation
Two main species of honeysuckle flowers are Lonicera japonica Flos (Jinyinhua) and Lonicera Flos (Shanyinhua) [18]. The stems of honeysuckle are also commonly used as medicine [6]. All of the flowers and stems of honeysuckle belong to the family of Caprifoliaceae [19] and contain similar chemical constituents [6], but they cannot be used interchangeably. With the help of Hubei Provincial Hospital of Traditional Chinese Medicine, six common honeysuckle samples with permit number 20160077 were purposefully selected for this study. These are flowers of honeysuckle: Lonicera japonica Flos (LJF, Pingyi County, Shandong province, China) and Lonicera Flos (LF, Longshan County, Hunan province, China), stems of Lonicera japonica: Lonicera japonica Caulis (LJC, Pingyi County, Shandong province, China), and stems of Lonicera: Lonicera Caulis (LC, Longshan County, Hunan province, China). In addition, LJF from Fengqiu County, Henan province, China and LF from Enshi County, Hubei province, China were also complemented for PCA to compare the differences between origins. All samples were packed in heat-seal vacuum aluminium foil bags for every 100 g portion sand stored in a 5˚C refrigerator. The aluminium foil bag can isolate the external humidity, and all samples were used within one week after repacking.

HS-SPME procedure
The HS-SPME procedure was conducted as described in our previous publication [20]. Two different sample preparation methods, namely dry and brewed, were applied in this study. In the dry method, 0.200 g of LJF (Shandong or Henan), LF (Hunan or Hubei), LC, or LJC was weighed (d = 0.001 g and max 620 g, Mettler Toledo, America) and added directly in a glass headspace vial with a PTFE-coated silicone septum (Agilent, California, USA). The brewed honeysuckle was prepared by adding 20.000 g of honeysuckle in 200.000 g of 80˚C Ultrapure water (Human Corporation Arioso Power Series, Seoul, South Korea). The optimal brewing conditions were determined to be 80˚C and 30 min (results not shown here). After brewing, the mixture was filtered through a metal sieve and chilled in an ice bath for 10 min. The cooled filtrate (2.000 g) was then transferred into a headspace vial. A Supelco 85 μm Carboxen/Polydimethylsiloxane (CAR/PDMS) fibre (Pennsylvania, USA) was used to extract volatiles from headspace vial at designated extraction temperatures (40, 60, and 80˚C) and extraction times (5, 30, and 60 min). Finally, the fibre was inserted in the GC injector for the subsequent GC analysis.

SAFE procedure
Brewed honeysuckle was prepared similarly as described above but in this case 60.000 g of sample was added in 600.0 g of 80˚C Ultrapure water was weighed by the balance (d = 0.1 g and max 8200). After brewing at 80˚C for 30 min, the mixture was filtered through a metal sieve and chilled in an ice bath for 10 min. The chilled filtrate (300 mL) was then transferred into a 500 mL beaker. An internal standard solution (0.060 g) was added to the filtrate and stirred for 5 min. The internal standard solution was prepared as follows: 0.300 g butyl butyryl lactate (VWR, Pennyslvania, USA), which was found not to coelute with other volatile compounds in honeysuckle in both MS and FID chromatograms, was added in 250 mL volumetric flask, then dilute to volume with ethanol (VWR, Pennyslvania, USA). The formula for calculation of the concentration of each compound was shown in Eq (1). The extraction conditions of SAFE was adapted from our previous work, with minor modifications [21]. The extraction conditions of SAFE (Glasbläserei Bahr, Germany) were 5 × 10 −6 mbar, the pressure was maintained by Edwards PFPE RV3 rotary vane pump (West Sussex, UK) and Edwards Diffstak diffusion pump (West Sussex, UK), and the water bath temperature was 40˚C.
Peak area of internal standard concentration of internal standard ¼ Peak area of each compound concentration of corresponding compound ð1Þ

GC-MS/FID analysis
Analytes quantitation and identification analysis were conducted by Agilent 7890B GC equipped with a flame ionisation detector (FID) and Agilent 5977B mass selective detector (MSD) (California, USA). CTC CombiPAL autosampler (Zwingen, Switzerland) was employed to insert samples into GC injector. GC column was Agilent HP-INNOWax column (60 m × 250 μm × 0.25 μm) (Woodbridge, USA). The GC conditions used were injector temperature 250˚C; splitless mode; helium carrier gas; column flow rate 1.2 ml/min; FID temperature was 300˚C; EI mode was 70 eV. The temperature gradient in the GC oven was 50˚C for 5 minutes, increased to 240˚C at rate of 5˚C/min, and held at 240˚C for 40 minutes. In-house and NIST 14 MS libraries (Connecticut, USA) were applied to identify the eluted compounds by matching the mass spectra, and further confirmed with the linear retention indices (LRI) of in-house standard compounds. LRI values of the HP-INNOWax column were calculated by a mixture of Supelco C7-40 alkanes (VWR, Pennsylvania, USA). Alkane standard and pure standards were run under the same conditions with the sample. All experiments were carried out in triplicate, and the results were reported as mean values of the peak area of each compound extracted by HS-SPME and mean values of concentration of each compound extracted by SAFE.

AEDA
For the AEDA analysis, the SAFE concentrates of LJF, LF and LJC were diluted stepwise with dichloromethane (VWR; Pennyslvania, USA) at 5 n (n = 0, 1, 2, 3, and so on). Each dilution was sniffed and evaluated via GC-O by four experienced flavourists (2 male and 2 female; aged 26-50). Gas chromatograph-olfactometry (GC-O) conditions were referred from our previous study [22]. All GC-O panellists had experience in conducting GC-O analyses before this study. The odor of each compound was described by these panellists. The strength of each compound was represented by their flavour dilution factor (FD-factor), which was the highest dilution at which nothing could be smelled at the GC-O port. Compounds corresponding to each odor were identified by injection of pure standards under the same GC-O condition.,

Data processing and statistical analysis
Student's t-test was performed to test the significance level of the difference between samples at two different conditions. Data of key aroma compounds obtained from AEDA was processed by PCA in the R programming language. The data was processed and transformed with data.table and magrittr packages and visualized them with ggplot2, ggbiplot, and patchwork packages.

The selection of HS-SPME extraction conditions
It is critical to select the extraction conditions (temperature and time) for natural materials. That is because high temperature and long-time extraction could destroy their volatile compounds, while lower temperatures and shorter extraction times could lead to poor sensitivity and recovery [23,24]. Therefore, different extraction temperatures (40, 60, and 80˚C) and extraction times (5, 30, and 60 min) were applied to study the effects of extraction conditions on the release of volatile compounds from honeysuckle (both dry and brewed).
As reported in the literature [25][26][27][28], hexanal, cis-3-hexenol, acetic acid, benzyl alcohol, and β-ionone were important compounds in honeysuckle. Hence, they were chosen as indicators for the effect of extraction temperatures on both dry and brewed LJF. In Fig 1(A), the peak area of hexanal, acetic acid, and β-ionone increased with the increase of temperature but decreased after 60˚C. Other aldehydes (e.g. 2-butenal, heptanal, trans-2-hexenal, and benzaldehyde) exhibited a similar trend. For cis-3-hexenol and benzyl alcohol, an increase in temperature resulted in the higher peak areas, which was observed for other alcohols such as geraniol, octanol, and 2-phenylethyl alcohol. Hence, alcohols were easier to be detected at the higher temperatures in dry honeysuckle.
The peak areas of the selected compounds after brewing are shown in Fig 1(B). Hexanal and other aldehydes such as 2-butenal, heptanal, trans-2-hexenal, and benzaldehyde showed an inverse trend compared to dry LJF. Hence, aldehydes were a little bit harder to be detected at a higher temperature in brewed honeysuckle. When the temperature was gradually increased from 40˚C to 80˚C, the peak areas of the most alcohol compounds showed high significant increase, except cis-3-hexenol, which reached the greatest peak area at 60˚C. cis-3-Hexenol appeared to stop rising at 60˚C, accompanied by increments in geraniol and β-ionone in samples after brewing, which is similar to the previous study [29], which may be the result of thermally-related reactions. The peak areas of acetic acid and β-ionone had the same trends as the peak areas increase when the extraction temperature was above 60˚C. Compared to dry honeysuckle extraction, acetic acid was more difficult to be isolated in brewed honeysuckle. Fig 2 shows the peak areas of major volatile compounds in dry and brewed LJF at various extraction times at 80˚C. In Fig 2(A), hexanal, cis-3-hexenol, and acetic acid were found to have the highest peak areas at 30 min, while benzyl alcohol and β-ionone recorded their respective highest peak areas at 60 min in dry LJF. For brewed LJF (Fig 2(B)), an extraction time of 30 min resulted in the greatest peak areas for cis-3-hexenol, acetic acid, and β-ionone. The peak area of benzyl alcohol was the highest with a 60 min extraction time, similar to dry LJF. Hexanal exhibited a unique trend. When the extraction time increased from 5 min to 30 min, the peak area of hexanal significantly decreased (p<0.001), and as the extraction time increased from 30 min to 60 min, the peak area of hexanal significantly increased (p<0.001). This phenomenon may be due to the hydration of hexanal and it could be fully extracted at low temperature [21].
The trends of each compound in dry LJC and LJC after brewing were similar to dry LJF and LJF after brewing. Hence, the results of the LJC were not shown in this study. Most of the volatile compounds in dry and brewed honeysuckle generally increased with the increase of temperature and had the greatest peak area at 30 min. Therefore, 80˚C and 30 min were chosen as the extraction conditions both in dry and brewed samples for further analysis.

Volatile profiles of different species and parts of honeysuckle by HS-SPME
Following the selection of HS-SPME extraction conditions, dry and brewed LJF, LF, LJC, and LC were analysed by GC-MS/FID under HS-SPME conditions of 80˚C and 30 min. One hundred and two compounds were tentatively identified using HS-SPME-GC-MS and shown in Fig 3. Higher volatile compounds were preferentially found, such as some aldehydes, limonene, γ-terpinene and terpinolene. The results are shown in S1 Table. The most abundant compounds in dry LJF were 2-butenal, hexanal, hexanol, cis-3-hexenol, acetic acid, benzaldehyde, octanol, benzyl alcohol, and 2-phenylethyl alcohol, etc., and these compounds have also been reported by Wang et al. [30]. The main compounds in brewed LJF were almost the same as those in dry LJF. However, the abundances of the same compounds were different in brewed and dry LJF. For example, the peak areas of aldehydes and acetic acid in brewed LJF were lower while alcohol compounds were greater when compared to dry LJF. The abundance differences were likely due to the different sample matrix. The changes of volatile compounds of LF were similar to LJF. Although hexadecenoic acid and octadecanoic acid were reported to be the quantitatively highest compounds in Lonicera Flos [31], they were not detected in our study. This might be due to the different extraction methods, such as microwave, hydro distillation, and ultrasound, it used. These methods cause thermal reactions or decomposition during extraction. Some terpenes (e.g. α-copaene, α-cubebene, γ-muurolene, and α-muurolene) were detected in dry LJC and LC but not in brewed stem and flower samples. Until now, there have been few studies on volatile compounds of honeysuckle, but they only focused on one species such as LJF or LF [25,30]. Few studies have been conducted to differentiate the volatile compounds in honeysuckle flower of two species, or in the different parts of a single species [6,32]. Hence, volatile compounds in various honeysuckles and different status may provide different aroma spectrum when they were added in food products.

Volatile profiles of different species and parts of honeysuckle extracted by SAFE
LJF, LF, LJC, and LC were isolated at 40˚C water bath by SAFE. S2 Table listed one hundred and eight compounds across all honeysuckle species, such as hexanal, 1-octen-3-ol, benzaldehyde, and eugenol, and these compounds were reported by literature [32]. SAFE was a complementary extraction method to HS-SPME due to its success in extracting higher polar compounds, higher molecular weight, and less volatile compounds such as eugenol, decanoic acid, and vanillin. SAFE is a modified distillation technique conducted under low-temperature and high-vacuum conditions, which aims to reduce the degradation of heat-sensitive volatiles and generation of artifacts as compared to other extraction methods like simultaneous distillation extraction (SDE) and hydro-distillation. When the extraction is performed using SDE, many linalool derivatives such as linalool oxides (furanoid and pyranoid) [25] as well as esters (bornyl actate, methyl hexadecanoate and methyl linolenate) [33] were detected. All these volatiles might be the result of thermal reactions during the SDE extraction process. Volatile compounds with lower boiling points such as pentanal, hexanal, trans-2-pentenal, and heptanal could not be detected by hydro-distillation [34] due to volatilisation during extraction. Therefore, the volatile compounds isolated by SAFE minimises the modification of volatiles in honeysuckle to obtain a more representative aroma extract.

Principal component analysis
Volatile studies often result in large, complicated datasets [35]. PCA is often used as a statistical tool to improve the efficiency of data analysis. In this study, PCA was conducted to pinpoint the main areas of variation among different species, parts and origins of honeysuckle. PCA could also suggest specific volatile features that contributed most significantly to the differences observed between honeysuckle samples. In the present study, the key odorants that were identified by GC-O and contributed to the aroma of honeysuckle were selected for comparison by PCA instead of the total volatile composition by GC-MS/FID. This allowed for a more streamlined analysis of various honeysuckles by eliminating volatile compounds that were not confirmed to be significant aroma contributors in honeysuckle. The data collected from dry and brewed LJF (Shandong and Henan province, China), LF (Hunan and Hubei province, China), LJC, and LC extracted by HS-SPME and SAFE are shown in Fig 4.  Fig 4(A) shows the biplot of key odorants in various honeysuckle extracted by HS-SPME-GC/MS without brewing. In the plot, PC 1 and 2 accounted for 45.6% and 27.0% of variations, respectively. Heptanol (11), 1-nonanol (20), and furfural (12) contributed greatly to the separation of LF from other species and parts of honeysuckle. Both LF samples extracted by HS-SPME contained more abundant amounts of these compounds regardless of their origins, although 1-nonanol (20) had a relatively greater peak area in LF Hunan. Similarly, γ-nonanolactone (32), 2-methoxy-4-vinyl phenol (34), and vanillin (36) were found to determine the separation of LJC on the positive area of PC 1 and negative area of PC 2 in the plot. In Fig 4  (A), the plot shows that the two LJF samples were clustered together, despite having different origins. A similar observation was seen in the two LF samples. Hence, the origin likely had a smaller impact on the differences in volatile compounds than the species. On the other hand, LJC and LC were separated from each other in the plot.

Conclusion
In this research, it was found that HS-SPME was able to extract volatile aldehydes more efficiently than SAFE, with larger peak areas obtained in the dry flowers of honeysuckle than in the brewed samples. With the help of SAFE, volatile compounds were minimally modified, and the compounds of lower volatility (e.g., eugenol, decanoic acid, and vanillin) were significantly enhanced. Based on the AEDA results, 36 key odorants were identified across LJF, LF and LJC SAFE extracts. Compounds with the highest FD-factors were 4-ethyl phenol, decanoic acid, 3-methyl-2-butenal, β-ionone, γ-octalactone, and vanillin, and the concentrations of key odorants among different honeysuckle species and parts were noted to be quite varied. PCA classified various honeysuckle extracted by HS-SPME and SAFE by using key odorants, and clustering was observed for flowers of the same species regardless of origins. The results concluded that the key odorants may potentially be used to discriminate honeysuckle by species. Therefore, this systematic analytical approach is important for determining the volatile compounds of various honeysuckles and would definitely provide the basis for extending the application of honeysuckle in food products.
Supporting information S1


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Effect of Fungal Extracts on Bacillus thuringiensis Serotypes Causing Flacherie Disease in Mulberry Silkworm, Bombyx mori L

Diseases are the most important limiting factor affecting silkworm, Bombyx mori L., known for the production of silk cocoons are generally affected by viral, fungal and protozoan pathogens among which bacterial pathogens alone cause cocoon loss about 7.5 to 20.0 percent (Selvakumar, 2013). The major fact responsible for bacterial flacherie was the rearing conditions, rise in temperature and humidity in rearing place, insufficient bed spacing leads to dysfunction of alimentary canal which encourages flacherie (Nataraju et al., 2005). Bacillus thuringiensis (Bt) is one of the causative agents for flacherie disease and the impact was high in silkworm larvae (Nishiit sutsuj et al., 1979). Bt is a Gram positive spore forming bacterium characterized by the formation of parasporal inclusions during sporulation (Ohba, 1996). After being ingested by the larvae, the parasporal inclusions are dissolved in larval midgut juice and release protoxins. The International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 2 (2020) Journal homepage: http://www.ijcmas.com

Introduction
Diseases are the most important limiting factor affecting silkworm, Bombyx mori L., known for the production of silk cocoons are generally affected by viral, fungal and protozoan pathogens among which bacterial pathogens alone cause cocoon loss about 7.5 to 20.0 percent (Selvakumar, 2013). The major fact responsible for bacterial flacherie was the rearing conditions, rise in temperature and humidity in rearing place, insufficient bed spacing leads to dysfunction of alimentary canal which encourages flacherie (Nataraju et al., 2005). Bacillus thuringiensis (Bt) is one of the causative agents for flacherie disease and the impact was high in silkworm larvae (Nishiit sutsuj et al., 1979). Bt is a Gram positive spore forming bacterium characterized by the formation of parasporal inclusions during sporulation (Ohba, 1996). After being ingested by the larvae, the parasporal inclusions are dissolved in larval midgut juice and release protoxins. The

ISSN: 2319-7706 Volume 9 Number 2 (2020)
Journal homepage: http://www.ijcmas.com Antibacterial activity of six different species of fungal extracts viz., Aspergillus ochraceous, Paecilomyces variotii, Schizophyllum sp., Epicoccum sp., Metarhizium sp., Aspergillus niger were evaluated against ten serotypes of Bacillus thuringiensis to develop new bioactive secondary metabolites from the microbial sources. Fungal crude extracts were isolated by ethyl acetate solvent extraction method and product yields varied from 0.150g to 0.538g. Thin Layer Chromatography was carried out for detecting the small molecule compounds present in the crude extracts. Antibacterial activities of fungal extracts were analyzed by disc diffusion method. All ten B.t serotypes were inhibited by six fungal extracts with zone of inhibition ranging from 12 ± 1.73 to 36 ± 1.53. Among the six extracts Paecilomyces variotii showed highest inhibition of 36 mm against B.t sotto. In vitro and in vivo studies conducted for the effect of extracts with different concentration and exposure duration reveals that effective against all serotypes. The results suggested that the Microbial derived extracts could be a new source of alternative in developing an effective bioactive molecule for the management of silkworm bacterial diseases. activated toxins interact with the larval epithelial membrane and induce pore formation in the membrane, which ultimately leads to insect death (Gill et al., 1992). Larvae affected by Bt lose their appetite, undergo convulsions and their bodies become stretched and cracked. The cadavers gradually become brown to black-brown and finally when rotting they turn black with fowling smell (Aruga, 1994). Researchers have come out with different sources of bioactive compounds/crude extracts from plants to prevent the silkworm bacterial pathogens (Selvakumar et al., 2001;Manimegalai and Chandramohan, 2005;Priyadharshini, 2008) but limited attempts were made for the use of microbial metabolites. In the present study, the efficacy of fungal extracts controlling the ten serotypes of Bt causing flacherie disease in mulberry silkworm was evaluated to develop a effective bioactive molecules for the control of disease.

Preparation of fungal extract
Fungal strains were grown in PDA culture medium at 28 °C for 5 days after that cultures were cut into solid discs (1cm 2 ) and inoculated into 1000 ml Potato dextrose broth in Erlenmeyer flasks. These cultures were grown with continuous shaking of 200 rpm at 25 ± 2 °C in shaking incubator for fifteen days. At the end of incubation, the fungal mycelium and the culture medium were separated by filtration using Whatmann filter No.3. The filtered supernatant was extracted thrice with equal volume of ethyl acetate. The combined organic phase was dried with anhydrous sodium sulphate and concentrated in reduced vacuum evaporator at 45°C. The crude extract dissolved in 10% dimethylsulfoxide (DMSO) and kept in 4 °C for further use.

Identification of small molecules
Thin Layer Chromatography technique was used for detecting the small molecule compounds present in the fungal crude extracts. The extract samples were spotted on silica plates and eluted in a TLC chamber with ethyl acetate and hexane as the mobile phase in the ratio 8:2. The plates were then air dried and visualized by exposing to iodine vapor.

In vitro antibacterial activity by disc diffusion
Antimicrobial susceptibility test was carried out by the disc diffusion method. Overnight incubated Bt cultures (1x10 8 spores/ml) spread on a Muller Hinton Agar plate. Whatman No.1 filter paper discs (6 mm diameter) dipped in the extract concentration of 50mg/ml solution and kept on inoculated plates. Ampicillin (10 mcg) was used as positive control. The plates inoculated with bacteria were made in triplicate and incubated at 32±1°C for 48 hours. The development of zone around the disc diameter was measured in millimeters. All data were analyzed through Microsoft Excel for Windows.

Efficacy of minimum antibacterial concentration
The effect of different concentrations of fungal product for minimum germicidal activity was tested in vitro against B. thuringiensis serotypes (1x10 8 cells/ml). These pathogens were suspended separately in different concentrations of fungal product (0.25 to 4%) for different durations 30 min, 1 hour and 2 hour at room temperature. After this incubation, samples were inoculated in Louria Bertani agar plate and incubated at 32±1°C for 48 hours. The experiment conducted in triplicate. The minimum bactericidal activity was determined by the lowest concentration of fungal product incubated with pathogens were completely killed.

In vivo activity of fungal extract
The antibacterial activity of the product with different concentrations tested in vivo against Bacillus thuringiensis serotypes (1x10 8 cells/ml). These pathogens suspended to the various concentrations of product and incubated at 25±2°C for different duration (30 min, 1 hour and 2 hour). After the treatment, samples centrifuged, washed and used for inoculation to determine the infectivity of the pathogens. 0.5 ml of this sample orally inoculated to third instar larvae.
In positive control, viable pathogens inoculated without treatment to the larvae and in negative control, without inoculation larvae reared for comparative study in three replications. All the batches were reared till spinning and allowed to spin the cocoons. Observations recorded for disease development and mortality. Treatment effects were analyzed to determine the differences between the treatment means of extract concentration.

Screening of small molecules
Antibacterial activities of the fungal extracts isolated from six different fungi were analyzed to derive high active biomolecules for treatment of Bt serotypes. The crude extract of fungal product yields varied from 0.150g to 0.538g. A range of yields among extracts was observed depending on the fungal material used.
The maximum extraction yield was obtained from Schizophyllum sp. with 0.538g and P. variotii exhibited the lowest extraction yield of 0.150g. Thin Layer Chromatography was carried out for detecting the small molecule compounds present in the crude extracts and 2 to 5 product spots were appeared in all fungal extract in the range of 0.2 to 0.84 retention factor (Rf) with ethyl acetate as mobile phase for the extract.

In vitro antibacterial activity
In vitro studies results showed that all six fungal extracts exhibited antibacterial activity in the range of 12 ± 1.73 to 36 ± 1.53 against Bt serotypes (Table 1), (Fig.2 & 3). The maximum activity of 36± 1.53 mm observed in P. variotii and 29 ± 1.00 in A. ochraceus against sotto and followed by 28 ± 0.58 mm in diameter against japonensis by Epicoccum sp. In particular, P. variotii and Epicoccum sp. showed above 20 mm inhibition against all serotypes of Bt except kenyae and tolworthi respectively. Among the six extracts, Metarhizium sp. exhibited lowest activities observed in all Bt bacteria except against sotto which was 25 ± 1.00 mm in diameter of growth inhibition. Ampicillin (10 mg) was used as positive control and results indicated that the extracts showed almost equivalent activity at low concentrations.      Table 2). In that 30 min exposure duration with the extract, 2 and 4% concentrations are effective and observed devoid of colonies. Up to 1% concentration was not effective and 10 to 50 bacterial colonies observed in 0.5, 1% and more than 300 colonies was observed in 0.025 % extract on the LB medium incubated for 48 hours. In 1 hour treatment similar trend was observed but number of colonies was less than the 30 min duration. In 2 hour duration, 1% extract onwards effective in all serotypes.

In vivo activity of extracts
An in vivo study reveals that 1% concentration of P. variotii extract effective for only 2 hour incubation, 2% and 4% extract was effective for all three exposed durations against Bt serotypes (Table 3). In positive control, without treatment larvae were infected by the pathogens but mortality rate was varied for all Bt serotypes. Bt morrsoni infected larvae died within 24 hours after inoculation of pathogen followed by japonensis, tolworthi, aizawai, sotto, alesti and less mortality rate was observed in kenyae, israelensis, kurstaki, thuringiensis with the influence of toxicity, temperature and/or food quality. Similar results reported by Selvakumar, (2013) stated that environmental factors have little impact on the mortality through toxicity and not infection by the bacteria.
Bacillus thuringiensis is a widely distributed facultative entomogenous bacterium well known for bio pesticide and employed extensively against insect pests in agriculture but they are pathogenic to the silkworm (Selvakumar et al., 1999). Mahmoud et al., (2019) reported that black seed and basil leaves extracts treated with Bacillus thuriengiensis infected larvae significantly decreased larval mortality in B. mori.
Similarly, aqueous extract of Thuja orientalis (Manimegalai and Chandramohan, 2005), leaf extract of Aegle marmelos and bark extracts of T. orientalis (Manimegalai et al., 2010) were effective in managing Bt infected B. mori. Similar observation was indicated by Mohanta et al., (2013) extracts of C. zeylenicum, C. longa, Z. officinale showed a strong antibacterial activity against Flacherie pathogens. The secondary metabolites have no apparent metabolic, physiologic and structural role in the producer, but have effects on other organisms function as biochemical defence (Jain et al., 2004). Zhang et al., (2011) isolated four active secondary metabolite (Cerebroside C, Cerebroside D, 2-Hydroxybenzyl alcohol, and 2-(4-Hydrroxypheny) ethanol) from the Paecilomyces sp. In the present study P. Variotii showed highest activity against all Bt pathogens and this might be presence of these compounds in the extract. Similar results reported by Oliveira et al., (2009) P. variotii extracts inhibited eight clinical isolates of entericococcs bacterial in the ranging from 25 to 35.25mm.
These results indicated that the possibility of the presence of highly active bioactive compounds/ secondary metabolites in the fungal crude extract. It is concluded that further studies are required to find out the bioactive compounds/ secondary metabolite responsible for the activity. Microbial derived products may be a source of alternative active compounds for control of silkworm diseases and it is a new approach in silkworm management.


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Genetic Divergence in Black Gram [Vigna mungo (L.) Hepper]

Black gram (Vigna mungo L. Hepper, 2n=22), popularly known as urd bean, Urid or Mash, is an important self-pollinating diploid grain legume (Naga et al., 2006) and belongs to the family Leguminosae and subfamily Papilionaceous. India is the primary centre of origin (Vavilov, 1926). It has been in cultivation from ancient times and is one of The present study entitled “Genetic Divergence in Black gram [Vigna mungo (L.) Hepper]” was conducted with 56 genotypes and two checks namely Pant U-31 and Pratap Urd-1during Kharif2019 at the Experimental farm, RCA, MPUAT, Udaipur. The genotypes were planted in augmented RBD design in four blocks. Mean squares due to genotypes were significant for plant height, number of branches per plant, number of pods per plant and pod length out of twelve characters as divulge from ANOVA. Present investigation results revealed that character seed yield per plant showed high GCV, heritability and genetic gain. Maximum heritability was found for pod length followed by plant height, number of branches per plant, number of pods per plant and seed yield per plant. While, maximum genetic gain was observed for seed yield per plant followed by number of pods per plant, number of branches per plant, plant height and biological yield. In general, PCV was higher than GCV for all the characters. Seed yield per plant was significant positively correlated at both genotypic as well as phenotypic levels with number of branches per plant, number of pods per plant, pod length, number of seed per pod, biological yield and harvest index. In cluster analysis, black gram genotypes were divided into VI clusters. Average inter cluster distance values were maximum between cluster V and VI (34.298). Cluster VI possessed genotypes with early flowering and maturity, plant height, number of branches per plant, pod length and harvest index From, the present investigation it can be concluded that genotypes KPU-129-104, RBU-38, KU16-89, KU-16-96, KPU-1098, TBU-2, KPU-11-37 appeared promising Presence of significant genetic variability and diversity among the tested genotypes of urdbean would be favorable for obtaining superior progenies in future breeding programme. K e y w o r d s

Introduction
Black gram (Vigna mungo L. Hepper, 2n=22), popularly known as urd bean, Urid or Mash, is an important self-pollinating diploid grain legume (Naga et al., 2006) and belongs to the family Leguminosae and subfamily Papilionaceous. India is the primary centre of origin (Vavilov, 1926). It has been in cultivation from ancient times and is one of The present study entitled "Genetic Divergence in Black gram [Vigna mungo (L.) Hepper]" was conducted with 56 genotypes and two checks namely Pant U-31 and Pratap Urd-1during Kharif-2019 at the Experimental farm, RCA, MPUAT, Udaipur. The genotypes were planted in augmented RBD design in four blocks. Mean squares due to genotypes were significant for plant height, number of branches per plant, number of pods per plant and pod length out of twelve characters as divulge from ANOVA. Present investigation results revealed that character seed yield per plant showed high GCV, heritability and genetic gain. Maximum heritability was found for pod length followed by plant height, number of branches per plant, number of pods per plant and seed yield per plant. While, maximum genetic gain was observed for seed yield per plant followed by number of pods per plant, number of branches per plant, plant height and biological yield. In general, PCV was higher than GCV for all the characters. Seed yield per plant was significant positively correlated at both genotypic as well as phenotypic levels with number of branches per plant, number of pods per plant, pod length, number of seed per pod, biological yield and harvest index. In cluster analysis, black gram genotypes were divided into VI clusters. Average inter cluster distance values were maximum between cluster V and VI (34.298). Cluster VI possessed genotypes with early flowering and maturity, plant height, number of branches per plant, pod length and harvest index From, the present investigation it can be concluded that genotypes KPU-129-104, RBU-38, KU-16-89, KU-16-96, KPU-1098, TBU-2, KPU-11-37 appeared promising Presence of significant genetic variability and diversity among the tested genotypes of urdbean would be favorable for obtaining superior progenies in future breeding programme. the most highly prized pulse crops of India. It is an economically important grain legume crop in Asia, widely cultivated on marginal lands with low inputs during Kharif, Rabi and Summer seasons. It is also an excellent green manure and soil conservation crop. Dehusked cotyledon is used for the preparation of fermented foods such as idli, dosa, and nonfermented foods like cooked dal, hopper, papad and waries (spicy hollow balls) (Batra and Millner, 1974). It is highly nutritious and is recommended for diabetic patients. Whole black gram is a rich source of protein, fibre, several vitamins and essential minerals such as calcium and iron (Reddy et al., 1982).
Black gram is the fourth main pulse crop in India. Total pulse production in India is estimated to 24.51MT whereas total production of Urd bean is recorded as 3.28 MT (DES, 2017-18). In India, black gram is cultivated in 5.031 Mha with a total production of about 0.653M tonnes. It is mostly grown in Andhra Pradesh, Uttar Pradesh, Maharashtra, Madhya Pradesh, Tamil Nadu, Rajasthan, Odisha and Bihar.
Assessment of variability is an initial step in any breeding programme. Higher the diversity of the material, better are the chances of improvement, provided that heritability and genetic advance is more. The ultimate goal of any breeding programme is to get higher yield. Correlation and Path analysis assists in evaluating yield contributing characters for selection. D 2 analysis has been found most effective and therefore, widely used for the classification of parental lines.

Materials and Methods
The present investigation was carried out during the kharif season of 2019 at Experimental farm, Rajasthan College of Agriculture, Udaipur is located at 24 0 35'N latitude and 73 0 42'E longitude and located at an elevation of 582.17 meters above mean sea level. The climatic conditions of that area were subtropical condition with humid climate. The soil of experimental field was clay loam, deep, well drained, alluvial in origin and has fairly good moisture holding capacity. The experiment was conducted in augmented RBD design with experimental material consists of fifty-six genotypes along with two check varieties (Pant U-31, Pratap Urd-1) of black gram were collected from Agriculture Research Station, Kota. Each genotype was sown in two rows with the spacing of 30 cm row to row and 10 cm plant to plant. Standard package of practices were followed to raise the crop. Observations were recorded on randomly selected five competitive plants for plant height, number of branches per plant, number of pods per plant, pod length, number of seeds per pod, seed yield per plant, 100 seed-weight, biological yield per plant, harvest index, and protein content. Whereas observation for days to 50 per cent flowering and days to maturity were recorded on plot basis.
The pooled experimental data were used for statistical analysis by using MS EXCEL and OPSTAT online software. Analysis of variance by Federer (1956) for augmented RBD Genotypic coefficient of variation (GCV) and phenotypic coefficient of variation (PCV) by Burton (1952). Heritability by Burton and De-Vane (1953).Genetic Gain by Johnson et al., (1955). Since, experimental design was augmented therefore, diversity analysis was carried out using ward (1963) method using Euclidian distances and calculated through SPSS version 19.

Results and Discussion
Mean squares due to genotypes were significant for plant height, number of branches per plant, number of pods per plant and pod length out of twelve characters as divulge from ANOVA representing some amount of genetic variability among the genotypes under study. Genotypes revealed variation for different characters viz., days to 50 per cent flowering (37 to 43 days), days to maturity (79 to 83 days), plant height (28.32 to 43.39 cm), number of branches per plant (5.99 to 12.01), number of pods per plant (14.75 to 36.12), pod length (4.00 to 5.65 cm),number of seeds per pod (4.24 to 7.12), seed yield per plant (2.03 to 5.56 g), seed weight (3.70 to 5.38 g), biological yield per plant (8.15 to 18.59 g), harvest index (12.94 to 51.63 %) and protein content (18.37 to 27.97 %).

Table.3 Number of black gram genotypes in each Cluster
Correlation coefficient analysis gives significant mutual relationship among various characters and yield so that selection can be more efficient to attain the goal of genetic improvement of crop for a complex trait like yield. Galton (1889) gave the concept of correlation coefficient and later on it was elaborated by Fisher (1918) and Fisher and Yates (1938). The phenotypic and genotypic correlation coefficients of all the characters were worked-out as per Al-Jibouri et al., (1958). The study revealed that seed yield per plant showed significant positive correlation at both genotypic and phenotypic levels with number of branches per plant (rg= 0.56 ** , rp = 0.47 ** ), number of pods per plant (rg= 0.53 ** , rp = 0.42 ** ), pod length (rg= 0.40 ** , rp = 0.40 ** ), number of seed per pod (rg= 0.49 ** , rp = 0.58 ** ), biological yield (rg= 0.73 ** , rp = 0.63 ** ) and harvest index (rg= 0.92 ** , rp = 0.60 ** ) and only at phenotypic level with 100seed weight (rp = 0.28 * ). It exhibited significant negative correlation only at genotypic level with seed protein content (rg = -0.95 ** ) ( Table 2) Path analysis is defined as simple standard partial regression coefficient which splits the correlation coefficient into the measure of direct and indirect effects of a set of component characters on the chief dependent variable. Path analysis was initially advocated by Wright (1921) and was further elaborated for plant breeding by Dewey and Lu (1959). Since, in the present investigation, the mean sum of squares due to genotype was not significant for seed yield per plant. Therefore, path analysis was not carried out as dependent variable yield is not significantly variable among them.
Genetic diversity is an important pre-requisite in selecting parents for hybridization and evolving high yielding genotyping in any crop breeding programme. D 2 analysis has been found most effective and therefore, widely used for the classification of parental lines. The concept of D 2 analysis was initially developed by P.C. Mahalanobis in 1928 but the application of this technique for the assessment of genetic diversity in plant breeding was advised by Rao (1952). The number of genotypes varied in each cluster from one to twenty. Fifty-six genotypes were grouped into VI clusters on the basis of pragmatic distance between genotypes within a cluster as related to genotypes in other cluster in Table 3. The maximum intra cluster distance was observed in cluster III (6.39) followed by in cluster I (6.03), cluster II (5.65), cluster VI (5.55) and cluster IV (4.99). Maximum inter cluster distance was observed between cluster V and VI (34.298). Cluster VI possessed genotypes with early flowering and maturity, plant height, number of branches per plant, pod length and harvest index (Table  4). Similar results were reported by Bharti et al., (2014), Geethanjali et al., (2014), Hadimani et al., (2016), Kamannavar et al., (2016), Reddy et al., (2018), Vidhya et al., regarding maximum and minimum intra and inter cluster distances in black gram.The genotypes belonging to different clusters revealed divergence for different traits and could be successfully employed in hybridization programme for desirable transgressive segregants. Therefore, crosses between members of high cluster means coupled with high inter-cluster distances among them are likely to be more rewarding.


extfieldsofstudy: Medicine Biology

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Assessing inbred–hybrid relationships for developing drought‐tolerant provitamin A–quality protein maize hybrids

Abstract Drought‐tolerant early‐maturing maize (Zea mays L.) inbred lines with high levels of provitamin A (PVA) and quality protein (QPM) are urgently needed for development of superior hybrids to mitigate malnutrition and to intensify maize production and productivity in sub‐Saharan Africa (SSA). This study was designed to identify early‐maturing inbred lines with combined tolerance to drought, elevated tryptophan, and PVA contents; to examine inbred–hybrid relationships for tryptophan and PVA accumulation; and to select hybrids with outstanding grain yield (GY) performance. A total of 64 inbred lines and six checks, plus 96 hybrids and four checks, were evaluated under drought and well‐watered environments in Nigeria for 2 yr. Eighteen parental lines and 54 derived hybrids were assayed for tryptophan and PVA contents. Ten drought‐tolerant inbred lines with high tryptophan and elevated PVA levels were identified in the top 10 hybrid combinations across managed drought and well‐watered conditions. The inbred–hybrid relationship was significant for GY under each and across the two contrasting environments. Significant average heterosis was found for tryptophan and PVA under well‐watered conditions. This indicated that the selected inbred lines could be used for developing high‐yielding PVA‐QPM hybrids tolerant to drought stress in SSA. The 10 top‐performing PVA‐QPM hybrids identified are being extensively evaluated in different locations and subsequently in on‐farm trials for commercialization throughout SSA.

increased rate of drought occurrence in maize production zones of the subregion significantly contributes to low grain yield (GY), which rarely exceeds 1.7 t ha −1 annually (FAOSTAT, 2016). Moreover, maize is often cultivated by small-scale farmers who are unable to afford irrigation. Interventions from governments and non-governmental organizations to provide irrigation facilities for low-input farmers have not been sustainable due to cost implications. In view of this, producers should not rely on seasonal weather patterns to provide water to the crop because extended periods of drought can reduce yield. However, improvement in the genetic ability of maize to tolerate drought environments has been identified as the most effective and sustainable means to significantly increase genetic gains in GY from selection (Abdulmalik et al., 2017;Badu-Apraku, Ifie, Talabi, Obeng-Bio, & Asiedu, 2018;Edmeades, Bolaños, Chapman, Lafitte, & Bänziger, 1999) for both small-and large-scale farmers in SSA. For instance, Oyekunle and Badu-Apraku (2013) assessed the performance of 156 early-maturing normal endosperm maize inbreds and determined heterotic patterns of the lines as well as the association between hybrid performance and performance of the lines per se. The hybrids and their parental lines were screened in different trials under moisture stress (drought) and optimal moisture (well-watered) conditions. Higher mid-parent heterosis (MPH) and better parent heterosis for GY were observed in the well-watered conditions relative to the drought conditions. Significant positive correlations were found between MPH, better parent heterosis, and GY for the two contrasting soil moisture regimes. High-yielding and stableyielding drought-tolerant hybrids were identified for commercialization in West and Central Africa. The authors concluded that drought-tolerant maize hybrids were of crucial importance in ensuring sustainable maize production and productivity in SSA.
Protein is an important dietary requirement for humans. Due to inadequate levels of lysine and tryptophan in the endosperm, normal maize grain is deficient in protein. These two amino acids account for the differences between quality protein maize (QPM) and normal maize. The discovery of several mutants (opaque2-o2, floury2-fl2, opaque7 -o7, opaque6 -o6, and floury3 -fl3) of the opaque 2 gene, which resulted in reduced levels of zein fraction but elevated levels of other protein classes and essential amino acids (Ma & Nelson, 1975;McWhirter, 1971), triggered more research into the improvement of maize for its quality protein content. The recessive homozygous genotypes (o2o2) have considerably higher concentrations of tryptophan and lysine in relation to the heterozygotes (O2o2) or dominant homozygotes (O2O2) (Crow & Kermicle, 2002). The identification of opaque-2 modifiers (Opm) responsible for the hardness of the o2-induced soft endosperm led

Core Ideas
• Drought-tolerant PVA-QPM hybrids are invaluable for human nutrition. • Ten PVA-QPM lines with high heterosis selected under drought for hybrid production. • Strong inbred-hybrid relationships aid selection of superior PVA-QPM hybrids. • Ten outstanding drought-tolerant PVA-QPM hybrids identified for commercialization.
to the breakthrough of selecting desirable QPM genotypes with kernel opaqueness of 25-50% (Babu, Agrawal, Saha, & Gupta, 2015). The QPM contains more than double the tryptophan and lysine content of normal maize (Teklewold et al., 2015;Vivek, Krivanek, Palacios-Rojas, Twumasi-Afriyie, & Diallo, 2008). As a result, overdependence on normal maize by children without enough dietary protein could lead to childhood growth failure, including "kwashiorkor," a fatal syndrome resulting from stunted growth (Gunaratna, De Groote, Nestel, Pixley, & McCabe, 2010). The QPM can potentially provide about 90% protein quality as percentage of milk, compared with approximately 39% in normal maize (Vivek et al., 2008). Furthermore, humans do not synthesize vitamin A in the body and thus rely on external sources to meet dietary requirements. Deficiency of vitamin A could lead to serious health disorders, including night blindness in children under 5 yr (Imdad, Herzer, Mayo-Wilson, Yakoob, & Bhutta, 2010). Breeding for elevated levels of provitamin A (PVA) in maize (a major staple food in SSA) endosperm was therefore necessary to ensure that maize becomes a cheap and sustainable source of vitamin A for the millions of people in SSA who depend on the crop for their daily calories. Several studies have elucidated the breeding strategies for biofortifying maize with elevated levels of PVA, and interesting findings have been reported. For instance, Harjes et al. (2008) reported positive but weak correlation between PVA levels and orange kernel color. Similarly, Azmach, Gedil, Menkir, and Spillane (2013) observed weak or no correlations between orange kernel color and PVA. The implication of these results is that depending on orange kernel color per se to determine the PVA levels of maize genotypes could be unreliable (Menkir & Maziya-Dixon, 2004;Menkir, Liu, White, Maziya-Dixon, & Rocheford, 2008). It is therefore necessary to routinely conduct chemical analysis to determine the levels of PVA carotenoids in the development of hybrid maize varieties for commercialization. Extensive studies have also identified marker-assisted selection to accelerate the detection and validation of PVA candidate genes, including β-carotene hydroxylase1 (crtRB1), lycopene epsilon cyclase (LcyE), and phytoene synthase1 (PSY1), which are involved in the regulation of PVA accumulation in the PVA biosynthetic pathway (Babu, Rojas, Gao, Yan, & Pixley, 2013;Harjes et al., 2008;Obeng-Bio et al., 2019;Owens et al., 2014;Wong, Lambert, Wurtzel, & Rocheford, 2004;Wurtzel, Cuttriss, & Vallabhaneni, 2012;Yan, Kandianis, Harjes, & Bai, 2010). The alleles of the crtRB1 gene (crtRB1-5′TE and crtRB1-3′TE) have been found to be the most favorable for PVA carotenoid accumulation in both tropical and temperate maize Obeng-Bio et al., 2019). The PVA maize can potentially provide higher than the daily dietary requirement of about 15.0 μg g −1 dry weight (DW) PVA for human beings relative to approximately 2.0 μg g −1 DW found in normal yellow maize cultivars .
The unique potential of maize calls for the need to develop superior drought-tolerant orange endosperm hybrids with enhanced levels of lysine and tryptophan as well as PVA not only to ensure food security but also to provide nutrient security, especially among children and women in SSA. To address this need, the International Institute of Tropical Agriculture Maize Improvement Program (IITA-MIP) has developed PVA inbred lines (from 2007 to 2015) that differ in their responses to limited soil moisture conditions, coupled with elevated levels of tryptophan and lysine. This has been necessary because successful performance of maize hybrids is largely associated with the inherent genetic potentials of the available outstanding parents (Betrán, Beck, Bänziger, & Edmeades, 2003). Information on inbred and hybrid genetic relationships is crucial to minimize time and cost involved in the production and evaluation of hybrids. Such information would offer the possibility to develop productive hybrids out of the myriads of hybrid combinations generated from the inbred lines available in a breeding program. Positive and significant correlation of GY between MPH of hybrid and parental lines has been reported (Betrán et al., 2003;Gissa, Zelleke, Labuschagne, Hussien, & Singh, 2007;Makumbi, Betràn, Bänziger, & Ribaut, 2011;Wegary, Vivek, & Labuschagne, 2013;Zeleke, 2015). Therefore, maize breeders need to consider the inbred-hybrid relationship while developing droughttolerant hybrids under drought and optimal growing conditions. However, there is a dearth of information on the parent-hybrid relationship in the available PVA-QPM inbred lines of the IITA-MIP.
In the present study, the PVA-QPM inbreds were screened with the aid of a light box to select desirable kernels (i.e., kernels that had deep orange color and endosperm opaqueness ranging from 25 to 50% for high PVA and tryptophan and lysine contents). It was therefore imperative that the PVA-QPM inbred lines be assessed under drought conditions and that the tryptophan and PVA contents of the inbred lines be determined with the use of chemical analysis as a basis for the development of PVA-QPM drought-tolerant hybrids in SSA. The objectives of the present study were (a) to identify PVA inbred lines combining drought tolerance and elevated levels of tryptophan, (b) to examine the relationship between the inbred lines and hybrids for increased levels of PVA and tryptophan under optimal growing environments, and (b) to select hybrids with outstanding GY performance across drought and optimal environments.

Field trials
The 70  was imposed by weekly application of 17 mm of sprinkler irrigation water until 25 d after planting, when the irrigation was suspended for the maize plants to depend on the residual soil moisture to complete growth and reproduction. For the trials under induced drought conditions, N-P-K (15-15-15) fertilizer was applied during planting at 60 kg ha −1 each for N, P, and K. Also, 30 kg ha −1 of N was applied as top-dressing at 3 wk after planting (WAP) using urea fertilizer. The inbred lines and hybrids were planted under wellwatered conditions at Ikenne during the 2016 and 2017 rainy seasons. Under optimal environments, N-P-K (15-15-15) fertilizer was applied to the inbred lines at 60 kg ha −1 each for N, P, and K immediately after thinning (2 WAP). At 4 WAP, top-dressing was carried out by applying urea fertilizer at 30 kg ha −1 of N. The experimental plots of inbred lines and hybrids were laid out using 7 by 10 and 10 by 10 lattice designs, respectively, each replicated twice. Inbred and hybrid fields were adjacent to each other. Each plot comprised a single 4-m-long row, with 0.75 m inter-row and 0.40 m intra-row spacing. Two plants were established per hill to obtain approximately 66,667 plants ha −1 . Preand postemergence herbicides (atrazine and gramozone, respectively) were applied at 5 l ha −1 to manage weeds as necessary.

Data collection
Based on individual plots for both inbred line and hybrid trials, data were taken for days to 50% anthesis (DA) and silking (DS) as well as plant and ear heights (PLHT, EHT). Plant aspect (PASP) and ear aspect (EASP) were rated on a scale of 1 to 9 (1 = excellent plants or ears; 9 = extremely poor plants or ears). The anthesis-silking interval (ASI) was estimated as the difference between DS and DA. At 70 d after planting, visual rating for leaf death (LD) was done for the trials conducted under induced drought using a 1-9 scale, where 1 indicates <10% of leaf death, and 9 indicates >90% of leaf death. The number of ears per plant was calculated as the number of ears harvested per plot divided by the number of all plants in the plot. Grain weight per plot was determined after shelling the harvested ears under drought environments. Moisture content of grain was measured with the aid of Kett moisture tester PM-450. Grain weight adjusted to 15% moisture content was used to estimate GY (kg ha −1 ) per plot. For the trials conducted under optimal growing conditions, 80% shelling percentage was assumed for computing GY based on ear weight adjusted to 15% moisture content.

Kernel samples production for quantification of tryptophan and provitamin A contents
Seventy-three genotypes including 54 hybrids, 18 inbred lines and a QPM standard check (Obatanpa) were selected to produce kernel samples for analysis of tryptophan and PVA contents. The 54 hybrids were selected from the 96 derived hybrids based on GY performance. The 18 inbred lines were parents involved in generating the 54 hybrids using the North Carolina design II mating arrangement. The genotypes were planted under well-watered conditions in 2018 at IITA-Ibadan (7 • 28′11.99″ N, 3 • 53′2.88″ E; 190 m asl) using 1-m-long single rows with 0.75 m between rows and 0.20 m within a row. One plant per hill was established to provide five plants per plot. Maize grain samples of hybrids and inbred lines were produced by self-pollination of individual plants in each plot. Ears harvested were dried to 12% moisture content at room temperature, and individual ears were shelled separately. Sixty kernels were taken from each ear (one ear per plant) to form a bulk representative sample of 300 kernels. Thereafter, 100 kernels were drawn from each genotype for PVA and tryptophan analyses using high-performance liquid chromatography (Howe & Tanumihardjo, 2006) and the colorimetric technique (Herbabdes & Bates, 1969), respectively, in the Chemical Laboratory of IITA, Ibadan, Nigeria. However, because Obatanpa is an openpollinated variety, the 100 kernels were drawn from a bulk of seeds harvested from an isolated field that consisted of over 1,000 plants. For quality protein levels, only tryptophan content was analyzed in endosperm flour. This is because tryptophan content has high correlation with that of lysine (r > .9) (Nurit, Tiessen, Pixley, & Palacios-Rojas, 2009). Moreover, tryptophan quantification is cheaper relative to lysine. Therefore, maize breeders of West and Central Africa usually estimate tryptophan content as a measure of the quality protein status of QPM genotypes, especially during the initial stages of the breeding schemes.

Statistical analysis
Data measured on the inbred lines and derived hybrids were used in ANOVA under each and across environments using the general linear procedure (PROC GLM) in Statistical Analysis Software, with a random statement and a test option (SAS, 2012). In the ANOVA, the location and year combinations constituted an environment, and the optimal and induced drought environments were regarded as research conditions. Environments, replicates within environments, and incomplete blocks within the interactions of replicates × environment were made random factors, whereas inbred lines and hybrids were made fixed factors in the statistical model. Significant differences among means were detected using standard error of difference (SED). Best linear unbiased predictions, also referred to as "predicted breeding values," were computed for PVA content of the inbred lines using the 'lmerTest' package in the R statistical software (R Core Team, 2013). The inbred lines were set as random factors, and the model was fitted using the 'lmer' function in R. Predicted breeding values for PVA content were computed using the 'ranef' function in R. Variance components due to genotype and phenotype of the inbred lines and hybrids were computed on genotype mean basis for the separate research conditions and across research conditions using the restricted maximum likelihood method. The variance component procedure (PROC Varcomp) implemented in SAS was applied to compute repeatability (R) for the measured traits as follows: where σ g 2 is the additive genetic variance (Hallauer, Carena, & Miranda-Filho, 2010), σ ge 2 is the genotype × environment interaction variance, σ 2 is the variance of experimental error, e is number of environments, and r is the replicates within an environment. Identification of drought-tolerant hybrids and inbreds was accomplished using the Multiple-trait base Index (M.I) proposed by Badu-Apraku, Fakorede, Oyekunle, and Akinwale (2011). The traits used in the selection index included GY, number of ears per plant (EPP), ASI, PASP, EASP, and LD under drought conditions as indicated in the following formula: Each trait was standardized using a SD of 1 and a mean of 0 to minimize the effects of different scales in the measurement of the different traits under drought conditions. Inbred lines and hybrids with positive M.I values were tolerant to drought, whereas those with negative values were susceptible to drought. In addition, ANOVA was performed for tryptophan and PVA data of the inbred lines and hybrids in SAS, and SED was used for the mean separation. The per se mid-parent value for parental lines was computed as the mean of the two inbred lines averaged for each research condition and across the two environments. Mid-parent heterosis (MPH) was estimated for the individual traits as follows: where F 1 is the hybrid mean, and MP is the mean of the parental inbred lines. Average MPH were separately estimated for the traits under optimal and drought conditions and across the two contrasting research conditions. The relationship between per se mid-parent values and MPH of corresponding hybrids were assessed for the traits under each and across the contrasting research conditions using the correlation procedure (PROC CORR) with the Spearman's rank method implemented in SAS. The difference between the mean of F 1 hybrids and that of mid-parents was tested for the measured traits by performing a t test (Wynne, Emery, & Rice, 1970) as follows; Note. ASI, anthesis-silking interval; DA, days to 50% anthesis; DS, days to 50% silking; EASP, ear aspect (1-9); Env, environment; EPP, ears per plant; GY, grain yield; LD, leaf death (1-9); PASP, plant aspect (1-9); PLHT, plant height; Rcond, research condition. * Significant at the .05 probability level. ** Significant at the .01 probability level.
where F 1ij represents the mean of the ijth F 1 cross, MP ij represents the mid parent for the ijth cross, and EMS represents the mean square of error.

RESULTS
Under drought conditions, ANOVA of the inbred trials revealed significant variation for environments (E) and inbred lines (G) for the measured traits except ASI and EASP for environment (Table 1). Inbred × environment interactions (GEI) were significant (p < .01 or p < .05) for the measured traits except LD. High repeatability (R) esti-mates were observed for the yield-related traits compared with that of GY under drought. The R estimates obtained for ASI and plant height among the inbred lines were high under drought. Furthermore, evaluation of the inbred lines under well-watered environments revealed significant (p < .05) variation for E, G, and GEI mean squares for the measured traits. The exceptions were E mean squares for ASI and E and GEI mean squares for PASP and EASP. Higher R estimates were found for most measured traits under well-watered conditions relative to their counterparts under drought conditions. Across drought and well-watered environments, significant variation was observed among E, research conditions (Rcond), inbred, inbred × Rcond, and GEI mean squares for the measured traits except Rcond mean squares for DA and DS. Repeatability estimates ranging from 12% for both DA and PASP to 64% for ASI were observed across research conditions. Analysis of variance of the hybrid data revealed significant variation among E, G, and GEI mean squares for most traits under each environment and across environments (Tables 2 and 3). The R values estimated for GY of hybrids were consistently high under moisture stress, under optimal moisture, and across environments. Forty-six percent of the inbred lines showed tolerance to drought, signalling their usefulness as an invaluable source of drought-tolerant alleles for the development of droughttolerant hybrids and synthetic varieties. Comparing the mean GY of the inbred lines for the drought trials with that under well-watered conditions revealed high variation in yield reductions from 16% for TZEIORQ 47 to 91.5% for TZEIORQ 32 (average, 59%) (Table 4). Generally, inbred lines with high and positive selection indices recorded lower ASI under drought environments.
Sixty-one percent of the hybrids were identified to be tolerant to drought on the basis of the drought-tolerant base index developed by the IITA-MIP. Ten inbred lines (TEIORQ 29, TEIORQ 24, TEIORQ 26, TEIORQ 20, TEIORQ 6, TEIORQ 47, TEIORQ 23, TEIORQ 7, TEIORQ 5, and TEIORQ 42) that combined elevated levels of PVA and tryptophan with drought tolerance were involved in the top 10 high-yielding PVA-QPM hybrids evaluated under drought (Table 5). Grain yield performance of the hybrids under drought conditions varied from 348 kg ha −1 for TZEIORQ 13 × TZEIORQ 15 to 5,291 kg ha −1 for TZEIORQ 29 × TZEIORQ 24 (mean, 3,592 kg ha −1 ). The highest-yielding PVA-QPM hybrid (TZEIORQ 29 × TZEIORQ 24) had the lowest percentage GY reduction of 2.3, whereas the lowest-yielding hybrid had the highest percentage GY reduction of 86. Moreover, analysis of data under drought environments generally revealed that hybrids that had reduced ASI and LD ratings and lower percentage GY reduction also had higher mean GY and positive selection indices. The best yielding PVA-QPM hybrid out-yielded the best commercial PVA-hybrid check (TZEI 124 × TZEI 25) by 32%.
About 90% of the inbred lines and all the hybrids assayed had more than 0.075% tryptophan per sample in whole grain ( Table 6). Seventeen of the 18 inbred lines assayed for tryptophan possessed elevated levels of tryptophan, of which 12 were drought tolerant. Obatanpa, the QPM standard check, had the highest content of tryptophan. In terms of tryptophan content, Obatanpa exceeded the best inbred (TZEIORQ 42) and the best hybrid (TZEIORQ 5 × TZEIORQ 47) by 37 and 23%, respectively. Generally, high contents of tryptophan were observed for the inbred lines and hybrids (Table 6). However, low to moderate levels of PVA (compared with the average daily dietary requirement of 15.0 μg g −1 DW for women and children under 5 yr) were found for both inbred lines and hybrids. Predicted breeding values (BVs) of PVA computed for the inbred lines ranged from −2.30 for TZEIORQ 47 to 3.76 for TZEIORQ 29. Inbred lines with positive BVs for PVA implied that they can accelerate gains from selection to increase PVA accumulation in derived hybrids. High repeatability (R) estimates of tryptophan and PVA were detected for the inbred lines and hybrids. This indicated that the use of recurrent selection procedures could improve the accumulation of PVA and tryptophan contents of populations undergoing recurrent selection programs. Wide variation in MPH of tryptophan was observed for the selected hybrids, ranging from 0.69% for TZEIORQ 7 × TZEIORQ 42 to 84% for TZEIORQ 5 × TZEIORQ 47. However, a relatively narrow range of MPH values of PVA (6% for TZEIORQ 5 × TZEIORQ 47 to 10% for TZEIORQ 29 × TZEIORQ 24) was detected for the hybrids (Table 6).
Significant positive correlations were observed between mid-parent values and the means of the corresponding hybrids for GY, PLHT, EASP, and LD under each environment and across environments (Table 7). Similarly, significant (p < .05 or p < .01) negative heterosis was observed for DA and DS under each environment and across environments. Significant (p < .05 or p < .01) average heterosis was also detected for tryptophan and PVA contents.

DISCUSSION
The highly significant differences in the inbred lines observed for all nine traits measured across the two contrasting research conditions implied the presence of genetic variation in the early-maturing PVA-QPM inbred lines, which is desirable for hybrid production. The significant inbred × environment interactions detected for the measured traits explained the inconsistent reactions of the inbred lines with respect to the traits measured under the contrasting environments and that evaluation in more environments would be necessary in identifying outstanding lines (Anley, 2013;Edmeades et al., 2013). The high repeatability (R) values estimated for the yield related traits, especially ASI and plant height, in relation to that of yield under managed drought conditions indicated that these traits could be relied on in addition to yield for the selection of outstanding drought-tolerant inbred lines (Bänziger, Edmeades, Beck, & Bellon, 2000 Note. ASI, anthesis-silking interval; DA, days to 50% anthesis; DS, days to 50% silking; EASP, ear aspect (1-9); Env, environment; EPP, ears per plant; GY, grain yield; PASP, plant aspect (1-9); PLHT, plant height; Rcond, research condition. * Significant at the .05 probability level. ** Significant at the .01 probability level. efficient pollination, fertilization, and seed set. Moreover, inheritance of good plant height (relatively taller plants) in hybrids indicates an advantage of producing enough assimilates for partitioning into sinks (grains), which could contribute to high GY. The high R estimate observed for ASI under low soil moisture environments confirmed the results of other researchers (Bänziger et al., 2000;Magorokosho & Tongoona, 2003;Santos et al., 2020), who determined that ASI is a predictable secondary trait for identifying drought-tolerant maize genotypes. It was striking to find higher R estimates for most traits under optimal growing conditions relative to their counterparts under drought. This result indicated the existence of maximum potentials of the inbreds in the absence of drought stress and that selecting for drought tolerance would be most successful when the experiments are conducted under drought conditions (Edmeades et al., 1999(Edmeades et al., , 2013 to expose the inherent responsive potentials of the genotypes to drought. Across drought and wellwatered environments, the variation due to environment, research conditions, inbred, inbred × research, and inbred × environment interactions were significant for most of the measured traits. This observation suggested that the two research conditions were very discriminating in identifying genetic variation in the set of inbreds evaluated. Relative to drought and well-watered conditions, low R estimates ranging from 12% for DA and PASP to 64% for ASI were observed across research conditions, suggesting that, when conducting drought experiments, ASI should be regarded as a key trait among the set of traits measured to identify outstanding drought-tolerant maize (Badu-Apraku et al., 2016;Bänziger et al., 2000), especially during early generation testing (Badu-Apraku, Oyekunle, Akinwale, & Aderounmu, 2013). The results revealed a wide range of yield reductions (16-91.52%), indicating that the response of the lines to the drought conditions was regulated by genetic factors because all the lines were exposed to the same level of moisture stress. This range was higher compared with the 40-90% reported by Menkir and Akintunde, (2001), an indication of wide genetic variability for drought tolerance within the PVA-QPM inbred lines. The significant variation observed for environment, genotype, and the genotype × environment interactions mean squares for most traits measured for the hybrids under each environment and across environments implied that there was high probability of identifying hybrids with superior performance in the set of 100 hybrids evaluated. High R values of GY were consistently estimated for the TA B L E 4 Performance of selected early provitamin A-quality protein maize inbred lines (best 10 and worst 5) plus best two checks under drought (Ikenne and Kadawa) and well-watered  hybrids for each and across environments, indicating that selection of drought-tolerant hybrids should be effective based on GY. Also, there is a higher possibility of transferring desirable characteristics of parental lines to hybrids. The practical implication of high R estimates is that highyielding hybrids under well-watered conditions would not suffer serious yield penalties under drought. However, this result disagrees with the findings of other researchers, who found GY to be lowly heritable especially under stress and that selecting for drought tolerance using GY alone may not be successful (Bänziger et al., 2000;Tuberosa, 2012). Sixty-one percent of the hybrids identified to be drought tolerant in the present study confirmed the reliability of the traits used in the multiple trait base index TA B L E 7 Correlation coefficients of mid-parent value of selected early maturing provitamin A-quality protein maize inbred lines and their hybrids, plus average heterosis under drought, optimal and across environments in Nigeria, 2016Nigeria, -2017Nigeria, /2018  . 6 9 ** --2 7 . 4 2 * a Not significant at .05 probability level; b Plant aspect (on a scale of 1-9), where 1 = excellent overall phenotypic appeal and 9 = poor overall phenotypic appeal. c Ear aspect (on a scale of 1-9), where 1 = clean, uniform, large, and well-filled ears, and 9 = rotten, variable, small, and partially filled ears. d Leaf death (on a scale of 1-9), where 1 = <10% overall dead leaf area, and 9 = >90% overall dead leaf area. * Significant at the .05 probability level. ** Significant at the .01 probability level. (Badu-Apraku et al., 2011). The 10 inbred lines that combined elevated levels of PVA and tryptophan with drought tolerance were also found to be the parents of the 10 highyielding PVA-QPM hybrids under drought environments. This observation demonstrated the significance of using drought-tolerant parents in the development of outstanding PVA-QPM hybrids and synthetic varieties to tolerate low soil moisture conditions (Betrán et al., 2003). The best yielding PVA-QPM hybrid (TZEIORQ 29 × TZEIORQ 24) appeared to have the lowest percentage GY reduction of 2.3, implying that this hybrid will continue to yield well under well-watered conditions. Similar results were obtained by Bänziger et al. (2000), who observed no yield penalties under well-watered conditions for the droughttolerant maize cultivars previously identified under managed drought environments. Generally, the hybrids that recorded high GY and positive selection indices also had reduced ASI, LD, and low percentage GY reductions.  , 2017). This implied that the 10 best-yielding, early-maturing PVA-QPM hybrids would be the most desirable for maize production in moisture-stressed areas of SSA. Extensive evaluation of these hybrids on-farm would be required to identify potential candidates for varietal release. Apart from the superior inherent ability of these hybrids to tolerate drought environments, they possess elevated levels of PVA and tryptophan as an added advantage over the existing commercial checks. Approximately 90% of the inbred lines and all the hybrids assayed for tryptophan concentrations had more than 0.075% in sample on a whole grain basis, suggesting that the inbreds and the hybrids met the quality criteria for classifying QPM genotypes from normal maize (Teklewold et al., 2015). The high levels of tryptophan suggest the existence of the opaque 2 genes and its modifiers for the accumulation of tryptophan and lysine concentrations in the endosperm of the inbred lines and the hybrids Crow & Kermicle, 2002). Similar inferences could be drawn for the source variety 2009 TZE-OR2-DT-STR-QPM, from which the inbred lines were extracted. The high contents of tryptophan observed for the inbred lines and hybrids suggested that QPM hybrids could be successfully developed from this set of inbred lines. However, the low to moderate levels of PVA found in both inbred lines and hybrids indicated that more breeding efforts are needed to successfully develop hybrids with the required PVA levels of ≥15.0 μg g −1 DW (Harjes et al., 2008;Ortiz-Monasterio et al., 2007). In view of this, predicted BVs were computed to identify parents that could facilitate gains for PVA accumulation in derived hybrids. This was necessary because reliance on orange kernel color per se for selecting high PVA maize genotype could be ineffective due to the positive but low correlation between orange kernel color and PVA levels (Azmach et al., 2013;Harjes et al., 2008;Menkir et al., 2008). Additionally, to accelerate progress in the development of superior hybrids with elevated levels of PVA, the IITA early and extra-early maize improvement program has concentrated efforts to consistently use the high-performance liquid chromatography method to select parental lines with high PVA levels for hybrid combinations. However, in the present study, the range of PVA values (3.47-12.10 μg g −1 DW) observed surpassed the 5.00-7.80 μg g −1 detected by Menkir et al. (2008) when 15 tropically adapted yellow maize inbred lines were evaluated. Also, PVA values ranging from 0.06 to 17.25 μg g −1 (mean, 5.87 μg g −1 ) have been reported by Azmach et al. (2013) using 130 inbred lines. The inbred lines that had high positive BVs for PVA in the present study (TZEIORQ 29,TZEIORQ 20,and TZEIORQ 24) could therefore make significant contributions to PVA accumulation in their progenies. This observation was confirmed by the highest PVA level of 10 μg g −1 recorded for TZEIORQ 29 × TZEIORQ 24, which ranked among the top 15 hybrids across the contrasting test environments. However, the differences in PVA levels of the top three droughttolerant hybrids were not significant. This implies that the three hybrids should be given equal opportunities for further evaluations as far as PVA levels are concerned. Also, the high repeatability (R) estimates of tryptophan and PVA detected for the inbred lines and hybrids were an indication of the possible rapid improvement in the two nutritional traits through the application of recurrent selection methods (Coors, 1999). The results also suggested that phenotypic selection based on dark orange kernel color could be effective (Chandler et al., 2013) in this set of hybrids. However, care must be taken in using orange kernel color to select maize genotypes with high PVA concentrations because over-reliance on this finding could be misleading (Azmach et al., 2013;Harjes et al., 2008;Menkir & Maxiya-Dixon, 2004;Menkir et al., 2008;Obeng-Bio et al., 2019).
The significant and positive correlations observed between the means of F 1 hybrids and the corresponding mid-parent means for GY, PLHT, EASP, and LD under each environment and across the two contrasting environments implied that early testing of the inbreds for the production of high-yielding, drought-tolerant hybrids based on these traits would be successful (Badu-Apraku, Akinwale, Franco, & Oyekunle, 2012). Similarly, the positive and significant inbred-hybrid correlation observed for PVA indicated that inbred lines with elevated levels of PVA could transmit high PVA accumulation ability to their hybrids, an indication of the existence of additive genetic effects controlling PVA accumulation in this set of inbred lines. Several researchers have found PVA accumulation in maize to be under the influence of additive genetic effects (Egesel, Wong, Lambert, & Rocheford, 2003;Menkir, Gedil, Tanumihardjo, Adepoju, & Bossey, 2014;Owens et al., 2014;Suwarno, 2012;Suwarno, Pixley, Palacios-Rojas, Kaeppler, & Babu, 2015), suggesting that application of recurrent selection methods should be effective (Coors, 1999). The nonsignificant correlation observed between inbred and hybrids for tryptophan content disagreed with previous findings. For instance, high tryptophan and lysine concentrations in hybrids were found to be primarily associated with the contributions of maternal inbred parents, suggesting that tryptophan and lysine contents were controlled by additive genetic effects specifically, maternal inheritance (Varadaraju & Joel, 2017). The differences in the results of the different studies may be due to the variation in the genetic materials used. Wide variation in MPH of tryptophan observed for the selected hybrids suggested that, although the hybrids were classified as QPM genotypes, significant differences existed in the ability of hybrids to accumulate elevated levels of tryptophan in their endosperm and that selection to upgrade the level of the trait would be successful. Despite the narrow range of MPH detected for PVA levels of the hybrids, the trait could be improved due to the positive PVA value observed. The significant and negative heterosis found for DA and DS for each and across the contrasting research conditions indicated that the F 1 hybrids matured relatively earlier compared with the parental lines and that the hybrids developed vigorous and taller plants, which translated into high GY. Ofori, Ofori, Obeng-Antwi, Tengan, and Badu-Apraku (2015) reported similar findings under optimal conditions. Significant average heterosis recorded for tryptophan and PVA contents implied that inbreds with relatively high concentrations of tryptophan and PVA could transmit the desirable attributes to their progenies (Betrán et al., 2003). Thus, superior hybrids with respect to drought tolerance as well as tryptophan and/or PVA accumulation could be developed using this set of inbred lines.
In conclusion, 10 outstanding drought-tolerant lines with high tryptophan and elevated PVA levels were involved in the best 10 hybrid combinations across managed drought and well-watered conditions. The selected inbred lines would be an invaluable resource for the development of maize hybrids possessing high tryptophan levels, elevated levels of PVA, and drought tolerance for farmers in the drought-prone production zones of SSA. The significant average heterosis obtained for plant and ear heights, DA, DS, and GY under drought indicated the high potential of developing outstanding drought-tolerant PVA-QPM hybrids from the 70 inbred lines studied. The 10 topperforming PVA-QPM hybrids identified in the present study should be widely assessed in advanced and on-farm trials and released for intensive maize production in SSA.

D ATA AVA I L A B I L I T Y S TAT E M E N T
Data pertaining to this study are available to authorized users at the International Institute of Tropical Agriculture (IITA) maize improvement program data repository.

A C K N O W L E D G M E N T S
This work was supported by the USAID through the West Africa Centre for Crop Improvement (WACCI), the Bill and Melinda Gates Foundation through the DTMA/STMA Projects (Grant OPP1134248), and the IITA. The authors thank Benjamin Annor and Gilbert Tchala Noudifoulè for their diverse contributions to this research.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.