Classification of Pigeonpea (Cajanus cajan (L.) Millsp.) Genotypes for Zinc Efficiency

Pigeonpea (Cajanus cajan (L.) Millsp.) is grown globally for its protein-rich seed. However, low availability of soil zinc (Zn) adversely affects the seed yield of pigeonpea. The present study was therefore conducted to assess the Zn efficiency of pigeonpea genotypes based on seed yield and seed Zn uptake efficiency. Field experiments were conducted at the Indian Council of Agricultural Research–Indian Institute of Soil Science, Bhopal, India with twenty different pigeonpea genotypes and two levels of Zn application under a split-plot design. The two levels of Zn were low (without application of Zn fertilizer) and high (with application of 20 kg Zn ha−1 (as ZnSO4∙7H2O) as basal soil application, in conjunction with three foliar sprays of 0.50% (w/v) ZnSO4∙7H2O aqueous solution) (with 0.25% lime as neutralizing agent) at flowering, pod formation, and pod filling stages). Application of Zn improved plant height, branches plant−1, pods plant−1, seeds pod−1, and 100 seed weight of pigeonpea genotypes differently. The mean seed yield, seed Zn concentration, and seed Zn uptake of the genotypes increased from 1.71 to 2.12 t ha−1, 32.4 to 43.0 mg kg−1, and 54.9 to 90.6 g ha−1, respectively, with application of Zn. The seed yield efficiency index (SYEI) and Zn uptake efficiency index (ZUEI) of pigeonpea genotypes varied from 67.0 to 92.5 and from 47.0 to 69.9, respectively. Based on SYEI and ZUEI, the genotypes were classified as efficient and responsive (Virsa Arhar-1, GT-1, GT-101, SKNP 05-05, BDN-2, AAUT 2007-04, BSMR 853, T 15-15, DT 23, Pusa 9), efficient and non-responsive (ICPL 87119, PKV Trombay), inefficient and responsive (AKT 8811, Hisar Paras), and inefficient and non-responsive (AAUT 2007-10, JKM 7, Hisar Manak, C 11, Hisar HO2-60, GAUT 93-17). The efficient and responsive genotypes are the most useful as they yield well under low soil Zn conditions and also respond to Zn fertilizer application. The inefficient and responsive genotypes could be utilized for plant breeding programs by plant breeders for identification and utilization of responsive traits.


Introduction
Pigeonpea (Cajanus cajan (L.) Millsp.), a protein rich legume crop, is cultivated in tropical and subtropical regions of the world. It is a vital grain legume crop in several countries of Asia, Africa, and Latin America. The largest share (≈75%) of global pigeonpea production comes

Results
The ANOVA for plant height, branches plant −1 , pods plant −1 , seeds pod −1 , 100 seed weight, seed yield, seed Zn concentration, and seed Zn uptake of pigeonpea genotypes indicated a significant effect of Zn treatments and pigeonpea genotypes in both years (Table 1). Therefore, the data for two years were pooled for statistical analysis. Table 1. Analyses of variance for plant height (m), branches plant −1 , pods plant −1 , seeds pod −1 , 100 seed weight (g), seed yield (t ha −1 ), seed zinc (Zn) concentration (mg kg −1 ), and seed Zn uptake (g ha −1 ) of pigeonpea genotypes for two years separately and pooled. F-ratio indicates ratio of two variances, LSD = least significant difference, CV = coefficient of variation, NS = not significant at p < 0.05, * = significant at p < 0.05. The weight of 100 seeds of pigeonpea genotypes varied from 8.00 to 11.3 g and from 8.20 to 13.1 g under low and high Zn supply, respectively (Table 3, Figure 2). On average, Hisar HO2-60 and AAUT 2007-04 had the lowest (8.10 g) and the highest (12.2 g) weight of 100 seeds, respectively. The seed yield of the genotypes varied from 1.24 to 2.54 t ha −1 and from 1.53 to 3.13 t ha −1 under low and high Zn supply, respectively ( Table 3). The mean seed yield of the genotypes increased from 1.71 to 2.12 t ha −1 with application of Zn. On average, PKV Trombay and ICPL 87119 recorded the lowest (1.39 t ha −1 ) and the highest (2.84 t ha −1 ) seed yield, respectively. Application of Zn enhanced the mean seed Zn concentration of the genotypes from 32.4 to 43.0 mg kg −1 (Figure 3). However, seed Zn concentration of the genotypes varied from 27.3 to 39.1 mg kg −1 and from 35.2 to 54.6 mg kg −1 under low and high Zn supply, respectively. On average, Pusa 9 and Hisar HO2-60 had the lowest (31.6 mg kg −1 ) and the highest (46.6 mg kg −1 ) seed Zn concentration, respectively. The mean value of Plants 2020, 9, 952 5 of 14 seed Zn uptake increased from 54.9 to 90.6 g ha −1 with Zn application (Figure 3). Seed Zn uptake of the pigeonpea genotypes varied from 39.3 to 75.8 g ha −1 and from 62.9 to 133 g ha −1 under low and high Zn supply, respectively. Plants 2020, 9, x FOR PEER REVIEW 5 of 15 kg −1 ) and the highest (46.6 mg kg −1 ) seed Zn concentration, respectively. The mean value of seed Zn uptake increased from 54.9 to 90.6 g ha −1 with Zn application (Figure 3). Seed Zn uptake of the pigeonpea genotypes varied from 39.3 to 75.8 g ha −1 and from 62.9 to 133 g ha −1 under low and high Zn supply, respectively.  Table 3. Hundred seed weight (g), seed yield (t ha −1 ), seed zinc (Zn) concentration (mg kg −1 ), and seed Zn uptake (g ha −1 ) of pigeon pea genotypes under low and high Zn treatments. SED = standard error of difference, LSD = least significant difference. NS = not significant at p < 0.05, * denotes significant at p < 0.05.   Table 3. Hundred seed weight (g), seed yield (t ha −1 ), seed zinc (Zn) concentration (mg kg −1 ), and seed Zn uptake (g ha −1 ) of pigeon pea genotypes under low and high Zn treatments. SED = standard error of difference, LSD = least significant difference. NS = not significant at p < 0.05, * denotes significant at p < 0.05.

Seed Yield Efficiency Index, Zn Uptake Efficiency Index and Classification of Pigeonpea Genotypes
The SYEI and ZUEI varied significantly among the pigeonpea genotypes (Table 5)

Discussion
Application of Zn increased plant height, branches plant −1 , pods plant −1 , seeds pod −1 , 100 seed weight, seed yield, seed Zn concentration, and seed Zn uptake of pigeonpea genotypes (Tables 2 and  3). On average, application of Zn enhanced plant height, branches plant −1 , pods plant −1 , seeds plant −1 , and 100 seed weight by 5. 24, 1.66, 49.4, 10.5, and 4.66%, respectively. Application of Zn increased mean seed yield, seed Zn concentration, and seed Zn uptake by 24.0, 32.7 and 65.0%, respectively. Other researchers also recorded a positive influence of Zn application on growth and yield

Discussion
Application of Zn increased plant height, branches plant −1 , pods plant −1 , seeds pod −1 , 100 seed weight, seed yield, seed Zn concentration, and seed Zn uptake of pigeonpea genotypes (Tables 2  and 3). On average, application of Zn enhanced plant height, branches plant −1 , pods plant −1 , seeds plant −1 , and 100 seed weight by 5.24, 1.66, 49.4, 10.5, and 4.66%, respectively. Application of Zn increased mean seed yield, seed Zn concentration, and seed Zn uptake by 24.0, 32.7 and 65.0%, respectively. Other researchers also recorded a positive influence of Zn application on growth and yield parameters of pigeonpea [22,23]. Similar to pigeonpea, green gram crop also responded positively to Zn application [24,25]. Zinc plays an important role in the growth and development of pigeonpea by contributing to the plant metabolism (synthesis of auxin, nucleic acid, and carbohydrate), enzyme activity, stomatal regulation, chlorophyll synthesis, pollen functions, and translocation of photosynthates [26]. Therefore, increase in Zn supply enhanced the growth and yield parameters of pigeonpea. Further, both soil and foliar application of Zn increased seed Zn concentration because of enhanced Zn translocation to seed from leaf and roots [27]. The responses of various pigeonpea genotypes to Zn application were different as shown by variations in enhancement as a percentage of growth and yield parameters. These variations were because of the existence of genetic divergence and phenotypic and genotypic variability among the genotypes [28,29].
The relationship among the growth and yield parameters of pigeonpea (Table 4) reveals that these parameters work in tandem. This finding is in agreement with the results recorded by David et al. [30] who studied 12 vegetable pigeonpea cultivars in eastern Kenya. Udensi et al. [31] also reported positive correlations of plant height with the number of branches and seed yield in soybean. The performance of crop plants depends upon the plant growth and development. Relations among the growth and yield parameters of pigeonpea were also reported by Saxena and Sharma [32], Sodavadiya et al. [33], and Pal et al. [34]. Plant architecture determines the performance of the plant by influencing light interception, photosynthesis, and translocation of photosynthates from source to sink [26]. The information pertaining to the relations of growth parameters with seed yield, seed Zn concentration, and seed Zn uptake could be used in selecting the genotypes for varietal development work.
The seed yield efficiency index and ZUEI were estimated (Table 5) to assess ZE of pigeonpea genotypes. These efficiency indices varied among genotypes. Similarly, ZE and manganese (Mn) efficiency (based on grain yield and Zn and Mn uptake efficiency) of wheat genotypes has also been reported by Singh et al. [35] and Jhanji et al. [21], respectively. The genetic and physiological traits of the genotypes govern ZE, which is also influenced by environmental conditions. The variations in ZE of the pigeonpea genotypes in the present study is corroborated by the differences in growth parameters such as plant height, branches plant −1 , pods plant −1 , seeds pod −1 , and 100 seed weight. Zinc efficient genotypes absorb and utilize Zn more effectively. Therefore, these genotypes could be grown in low soil Zn conditions for increased seed yield and seed Zn uptake to achieve food and nutritional security.
The genotypes DT 23 (92.5) and T 15-15 (92.5) had the highest SYEI and AAUT 2007-10 had the lowest SYEI (AAUT 2007-10). However, GT 101 had the highest (75.4) ZUEI and PKV Trombay had the lowest (47.0) ZUEI. Since both higher seed yield and increased seed Zn uptake are required traits, the pigeonpea genotypes were classified into four groups, namely, ER, IER, IENR, and ENR (Table 6, Figure 4), considering both SEEI and ZUEI. The ER genotypes (Virsa Arhar1, GT-1, GT-101, SKNP 05-05, BDN-2, AAUT 2007-04, BSMR 853, T 15-15, DT 23, and Pusa 9) yield well under low soil Zn conditions but also respond well to application of Zn fertilizer. The IER genotypes (AKT 8811 and Hisar Paras) produce low yield under low soil Zn conditions but respond to Zn fertilizer application. The IENR genotypes (AAUT 2007-10, JKM 7, Hisar Manak, C 11, Hisar HO2-60, and GAUT 93-17) neither yield well under low soil Zn conditions nor respond to Zn fertilizer application. The ENR genotypes (ICPL 87119 and PKV Trombay) yield well under low soil Zn conditions but do not respond to Zn fertilizer application. Both ER and ENR genotypes could be grown by farmers in low soil Zn conditions to obtain better seed yield. In contrast, both ER and IER genotypes could be utilized for agronomic Zn biofortification programs. Since IER genotypes do not yield well under low soil Zn conditions, these genotypes could not be considered by the farmers for cultivation. However, these genotypes could be utilized by plant breeders for plant breeding programs. Moreover, plant breeders could isolate and use the Zn efficiency and Zn responsive traits from the identified genotypes of pigeonpea for developing desired genotypes of pigeonpea and other crops.

Experimental Details
The experiments were carried out in a split-plot design with Zn application as the main-plot treatment and pigeonpea genotypes as sub-plot treatments. There were two levels (low and high) of Zn treatments and three replications. In low Zn treatment, no Zn fertilizer was applied. Under high Zn treatment, 20 kg Zn ha −1 (as ZnSO 4 ·7H 2 O) was applied as basal soil application in combination with three foliar applications of 0.50% (w/v) ZnSO 4 ·7H 2 O aqueous solution) (with 0.25% lime as neutralizing agent) at flowering, pod formation, and pod filling growth stages [35,[44][45][46], The pigeonpea genotypes were cultivated in plots of 6 × 5 m 2 area, with row to row spacing of 80 cm and plant to plant spacing of 20 cm. The plot of each genotype was separated from the plot of another genotype by 1 m. The crops were supplied with 40 kg of N (as urea) ha −1 , 80 kg of P 2 O 5 (as diammonium phosphate) ha −1 , 30 kg of K 2 O (as muriate of potash) ha −1 , and 40 kg of S (as bentonite S) ha −1 [47]. Hand weeding was carried out as and when required to keep the field weed free.

Recording Observations for Growth Parameters, Plant Sampling and Analysis
At physiological maturity, observations pertaining to growth parameters, such as plant height, branches (primary + secondary) plant −1 , and pods plant −1 were recorded, by averaging the values for 10 plants in each plot. Pods were harvested at physiological maturity and subsequently threshed after sun drying. The number of seeds pod −1 , 100 seed weight, and seed yield were recorded. For estimating seed Zn concentration, 10 pigeonpea plants were collected from each plot and seeds were separated by hand threshing. Seed samples were washed with double-distilled water three times and then oven dried at 75 • C for 48 h. Dried seed samples were ground in a stainless-steel mill (Micro-mill grinder, model −0210 × 60, Bel-Art-SP Scienceware) to <1 mm. Powdered seed samples were digested in a di-acid mixture (nitric acid and perchloric acid mixture in 9:4 ratio) [48]. The concentration of Zn in digested material was determined by atomic absorption spectrophotometer (Make and model: Varian AA 240 FS). The Zn uptake in pigeonpea seed, seed yield efficiency index (SYEI), and Zn uptake efficiency index (ZUEI) [49] were estimated as described below.
Zn uptake in pigeonpea seed (g ha −1 ) = Seed yield (t ha −1 ) × Seed Zn concentration (mg kg −1 ) (1) Seed yield efficiency index (SYEI) = Seed yield under low Zn Seed yield under high Zn × 100 Zn uptake efficiency index (ZUEI) = Seed Zn uptake under low Zn Seed Zn uptake under high Zn × 100 The pigeonpea genotypes were classified into four groups based on their SYEI and ZUEI values. The mean values of SYEI and ZUEI were used as criteria to classify the genotypes [21,35]. Group 1 consisted of efficient and responsive (ER) genotypes having higher efficiency indices than the mean values of SYEI and ZUEI. Group 2 consisted of efficient and non-responsive (ENR) genotypes having higher SYEI but lower ZUEI compared to their respective mean values. Group 3 consisted of inefficient and responsive (IER) with lower SYEI but higher ZUEI. Group 4 had inefficient and non-responsive (IENR) genotypes having lower SYEI and ZUEI than the mean values.

Statistical Analysis
The data were analyzed using analysis of variance (ANOVA) [50] to evaluate the differences between treatment means. The standard errors of differences (SED) were derived from the ANOVA table using the SAS 9.2 software package [51]. Because the least significant difference (LSD) has more power compared to other post hoc comparison methods [52,53], it was used for comparisons where F-probabilities were significant (p < 0.05). The year-wise analysis of data was carried out and then pooled for two years as the year-wise variations in data were found to be non-significant.

Conclusions
Increased Zn supply improved growth parameters, seed yield, seed Zn concentration, and seed Zn uptake of pigeonpea genotypes. Zinc efficiency measured in terms of seed yield efficiency and seed Zn uptake efficiency of genotypes differed significantly. The genotypes could be classified into four groups, namely, efficient and responsive, inefficient and responsive, inefficient and non-responsive, and efficient and non-responsive. Both efficient and responsive and efficient and non-responsive genotypes can be suggested for farmers for cultivation to obtain higher seed yield under low soil Zn conditions. The inefficient and responsive genotypes could be used for plant breeding programs for isolation of genes accountable for the response to Zn fertilizer application. The inefficient and non-responsive genotypes could be improved by plant breeding programs by incorporating efficiency and responsive traits. The genotypes of other important crops could be assessed for Zn efficiency for their classification into different groups and use by various stakeholders.