Evaluation of Newly Bred Zinc and Iron Enriched Rice Genotypes for Cultivation under Aerobic Condition using Genetic Parameters: Implication on Drought Management

This research focused on the evaluation of twelve newly bred rice genotypes with diverse morphological traits under aerobic cultivation to develop strategies for water conservation while maintaining grain productivity and quality. The experiment was conducted during the summer season of 2016, employing a Randomized Complete Block Design (RCBD) with wetland cultivation as a control. For analysis, various vegetative and reproductive traits namely, Plant height, Number of Tillers, Shoot Weight, Days to 50% flowering, Days to Maturity, Number of Productive Tillers, Panicle Length, Grain Weight, Length of the seed, Breadth of the seed, Ratio of Length and Breadth of the Seed, and Test weight of 100 seeds were recorded. Data on analysis of variance revealed the significant difference for days to flowering, maturity, plant height, panicle length, test weight, and grain yield under aerobic and wetland cultivation methods during the summer season, 2016, suggesting these traits are under high genetic control. Genetic parameters such as percentage of GCV, PCV, Heritability, and GAM were estimated across 13 parameters comprising vegetative and reproductive characters. The findings suggest a considerable amount of genetic variability for most traits, providing a foundation for the potential implementation of an effective selection procedure in breeding new rice varieties within the selected genotypes.


Introduction
Rice (Oryza sativa L.), is a crop for assuring nutrition and food security as a chief source of energy and stable food for half of the global population, majorly for Asian countries. 1Asia is the most significant contributor to global rice production, accounting for around 90% of the total output.Rice is indeed a versatile crop with a high degree of adaptability to cultivate in dry and wetland conditions in a wide range of latitudes and under various soil, climatic, and hydrological conditions. 2 The global average yield of irrigated rice is 5 t ha−1 and the variations in yield averages are based on factors like location and season in addition to the genetic contribution of various genotypes3.The yield of rice grain varies from 7-8 t ha−1 and 5 -6 t ha-1 and during dry and respectively. 4As the population continues to grow, there is an increase in the demand for rice, particularly in Asia and Africa. 5As the global population continues to grow in Asia, the need for increased rice production becomes crucial to ensure food security in addition to meeting the dietary needs of billions of people. 6 meet ever-increasing demand, it is important to scale up rice production either by increasing yield per unit area or expanding areas under cultivation. 7he intensive requirement of water for the cultivation of rice is a growing concern for many rice-growing countries.Although a number of Agro-practices are suggested to minimize the water requirement for cultivation, the development of newer and drought-hardy genotypes coupled with an evaluation under minimal water is one of the viable strategies proposed.More recently, the concept of cultivating rice under aerobic conditions is gaining larger attention as this method requires minimal water similar to that of other dryland crops.The aerobic method involves planting or seeding directly on the soil without prior establishment of a nursery. 8The development of rice varieties with superior grain yield under low input including limited water usage is of paramount importance in the current scenario of agriculture. 9In addition, productivity is one of the major objectives of rice breeding programs. 10entification of rice cultivars with improved agronomic traits is essential in rice breeding. 11enetic variation is the basis of plant breeding and provides a great array of genotypes as a source of genetic stock. 12To assess the performance of a genotype, reliable estimates for heritability are essentially required and this would provide the knowledge to predict the nature of the succeeding generation, to make an appropriate selection, and to assess the magnitude of genetic improvement through selection. 13,14This study was conducted to evaluate the performance of 12 genotypes of rice cultivars under two extreme cultivation methods (wetland and dry land) in the summer season of India.Analysis of variations followed by estimation of genetic variability using genetic parameters such as genetic coefficient variation (GCV), phenotypic coefficient variation (PCV), heritability at broad sense (h 2 ), mean percent of genetic advance (GAM) with respect to yield and its attributing traits were analyzed.The implication of the present study to develop breeding strategies for generating new hybrids for an aerobic method of cultivation as one of the strategies to minimize the water requirement for the cultivation of rice is discussed.

Selection of Genotypes
Seven elite rice lines, characterized with high iron and zinc along with the two checks and three parents were selected. 15,16The selected genotypes were generated by advancing the F2 generation through F8 by following the principles of single-seed descent followed by rapid generation from parents of Azucena and Moromutant (AM 1, AM 27, AM 65, AM 72, AM 94B, AM 143 B), Buddha X IR 64 (ARB 6) locally named as Anagha or ARB -6.The national yield check IR 64 and micronutrient check Chittimuthyalu was also included in the experiment (Table 1)

Experimental Area and Design
This study was carried out in the experimental field of Aerobic Rice Research Laboratory, Department of Plant Biotechnology, University of Agricultural Sciences, Gandhi Krishi Vignan Kendra.Bangalore.The experimental plot is located at the latitude of 12 o 58' North longitude, 77 o 35' East and altitude of 930 meters above mean sea level (MSL).Randomized Complete Block Design (RCBD) was laid-out with three replications in aerobic water regimes with a similar set of replications for wetland cultivation (a control) during summer season (Figure 1).

Preparation of Aerobic Condition
Aerobic rice cultivation involves growing rice in non-flooded or well-drained conditions with minimal water usage for irrigation.This method is contrasting with the traditional method, where the rice plant is submerged for three fourth of the period under cultivation, needing a huge quantum of water.In the aerobic method of rice cultivation, fields are not flooded throughout the cropping season and water is applied over the soil surface at regular intervals, similar to that of other dryland field crops. 8To construct a temporary structure to stimulate wetland or water-puddled conditions, a tank measuring 11m x 9m x 1m (length x width x height) was covered with waterproof tarpaulin and filled with soil the same of the aerobic plot.The tank was lined with PVC pipes to avoid the escape of water from the experimental tank.

Nutrition and Plant Protection Measures
The experimental plot was maintained based on the recommended package. 17Nitrogen was supplemented in the form of Urea at the basal dosage of 50% during sowing, 25% after 30 days after sowing (DAS) and 25% after 60 DAS for High Nitrogen Condition.Phosphorous (P) as single super phosphate (16% P2O5) was applied during basal dosage for High Phosphorous conditions.Potassium (K) was applied in the form of a Murate of Potash (60% K2O) in a Split of 50% during sowing and the rest 50% at 30 DAS.Measures were taken to control pest and disease by drenching soil with Chlorpyrifos (0.3 milliliters per liter of water) to control root grub.
A spray of Tricyclazole (0.6 grams per liter of water) was applied to avoid the emergence of blast disease.

Data Collection
Five plants were randomly selected from the center of replicated plots of each genotype.Observations on vegetative (plant height, number of tillers, shoot weight) and reproductive (days to 50% flowering, number of productive tillers, panicle length, grain weight, length of the seed, breadth of the seed, weight of the 100 seeds) parameters was carried out for statistical analysis for the proposed genetic parameters.The data was compiled using the mean value of the three replicates of phenotypic observation of each genotype.The data was subjected to Statistical Package for Agricultural Research (SPAR2.0). 18

Analysis of Variance
Analysis of variance for various traits recorded in aerobic and wetland conditions during the summer season was performed based on the standard methods. 19,20The significance of the experiment was arrived at by comparing the data with the standard values. 21The Standard Error of the Mean (SEM) 22 and the Critical Difference (CD) 23 were used to compare the mean of individual data.

Genetic Variance and Correlation Analysis
Genetic Variance (GV) and Phenotypic Variance (PV) were calculated based on the established method. 24,25The genetic parameters for yield and its attributing traits were determined and genotypic and phenotypic coefficient variation was computed for analysis. 26The percent of phenotypic coefficient variance (PCV) and genotypic coefficient variance (GCV) was calculated. 27The genetic advance was determined and the percent of mean of genetic advances was calculated. 28The Heritability (broad sense) was estimated for all the traits using the ratio of genotypic variance and phenotypic variance.The heritability percent was determined. 29The correlation analysis which shows the degree of association between the traits was determined by computing the phenotypic data set. 30

Analysis of Variance (ANOVA)
The analysis of variance for 13 quantitative characters under the aerobic cultivation method during summer showed a high degree of significance among the evaluated genotypes of rice for days to flowering (265.78),days to maturity (195.27),plant height (1338.94),grain weight (57.03) and panicle length (29.93).All 13 characters are highly significant among all the 12 genotypes.A similar observation was made with respect to the wetland method of cultivation, indicating that genotypic influences are dominating over the agronomic characters rather than the influences of the two methods of cultivation employed in this study.Therefore, the above quantitative characters are applicable for the selection and hybridization of significant traits on tested genotypes.Details of the results are presented in Table 2. a and 2. b.

Variability, Genetic Advance, and Heritability
Reproductive traits such as days to fifty percent flowering (DFF) and days to maturity (DM) were found to have low GCV and PCV values (> 10 %).Most of the parameters associated with quantitative traits of rice grain such as panicle length (PL), Length of the seed (SL), breadth of the seed (SB), length/breadth ratio of the seed (LB ratio), test weight (TW) was found to have moderate GCV and PCV (10-20%).High GCV and PCV (<20 %) were recorded for traits namely, plant height (PH), number of tillers (NT), number of productive tillers (NPT), shoot weight (SW), grain weight (GW) and biomass (BM).High heritability at broad sense (<60%) is observed in the traits such as days to 50% flowering (DFF), days to Maturity (DM), plant height (PH), number of tillers (NT), number of productive tillers (NPT), panicle length (PL), shoot weight (SW), grain weight (GW), biomass (BM), length of the seed (SL), breadth of the seed (SB), Ratio of Length and Breadth of the Seed (LB ratio), test weight of 100 seeds (TW) Low Genetic percent cent mean (10-20%) in days to maturity under aerobic condition during summer.advances as percent mean (>10%) were observed in traits namely, days to maturity (DFF) in wetland conditions during summer, and moderate Genetic advance as.

Discussion
Genetic improvement of rice is a continual process across the world due to the increase in demand for rice grain to meet the ever-increasing population.
Although molecular breeding become a viable tool for genetic improvement, conventional breeding combined with trait-associated selection with various genetic parameters is very fundamental in the rice breeding program.India is one of the major rice producers and the considerable number of rice varieties were exploited for commercial cultivation.In this study, seven of the genotypes are known to have high amount of Zinc and Iron. 31Therefore, evaluation of these genotypes for their performance with respect to several agronomic traits are important.In this study, the analysis of variance showed a very high significant difference among the genotypes evaluated across most of the traits as reported by a number of researchers. 11,35,36,37This indicates that rice genotypes are highly variable for several agronomic traits, facilitating scope for genetic improvement through various breeding strategies.
The detailed results on genetic parameters are presented (Table 3) High degree of genetic variability (genotypic and phenotypic) was found with most of the traits from low to high.Higher genotypic variability (GCV) and phenotypic variability (PCV) were recorded for most important agronomic traits namely, plant height (PH), number of tillers (NT), number of productive tillers (NPT), shoot weight (SW), grain weight (GW) and biomass (BM).GCV and PCV at more than 20% is considered as high, whereas values less than 10% are considered to be low and values between 10% and 20% being considered to be moderate38.According to this, most the traits have high to intermediate GCV and PCV for all the genotypes of rice evaluated in this study.Therefore, these traits could be used as the most probable genetic indicators during the process of selection and hybridization of elite genotypes in genetic improvement of rice varieties as suggested. 39e observed results on genetic variation clearly show that the PCV values are slightly higher than GCV values but the difference was not larger.For example, PCV & GCV values for DFF, DM, PH and NT, and NPT were respectively low under the wetland method of cultivation during summer.However, for certain of the traits such as GW and NT, the difference between PCV and GCV values is slightly higher with the dominance of PCV over GCV.Therefore, a higher difference between PVC and GVC could be an indication of environmental influence on phenotypic expression as previously reported in rice. 40However, the difference between genotypic and phenotypic coefficient of variation is very little for all studied traits.This result indicated that the influence of environment on the expression of most of the traits is very small and the present findings are supported by a number of previous works. 41,11,35,42oad sense heritability is classified as low (>60%), medium (30% to 60%) and high (>60%) based on the published report. 28All the traits included in the present study were found to have higher heritability among the selected genotypes of rice.It was reported that direct selection cannot be followed if the broad sense heritability is either lower or medium.However, a combination of broad sense heritability with other strongly inheritable genetic parameters can be taken during genetic evaluation. 28netic advance as percent of mean (GAM) is classified as low (10%) moderate (10%-20%) and high (>20%) [18].In this study, GAM is ranging from low (18.14 for DFF under wetland condition) to high (77.42 for GW under Aerobic condition) across the various traits.However, only a few traits are found to have higher GAM.For example, 60.82 for NPT under wetland condition, 62.94 for SW under aerobic condition, 68.35 for BM for aerobic condition and 77.42 for GW in aerobic condition.These traits are found to have GAM% more than 60% and can be used as genetically useful marker for utilization in rice breeding program of selected rice genotypes.Higher values of GAM for the above traits are appears to be governed by non-additive gene action.Therefore, these traits are highly useful selection procedure for breeding newer varieties of rice 42,43,44 In this study, we have demonstrated the successful cultivation of different genotypes of rice under minimal water usage in aerobic cultivation method and the amount of genetic variability among the selected genotypes of rice were determined.This study also opens up a new dimension of research on water use efficiency under drought prone areas for cultivation rice.As the selected genotypes are enriched with Zinc and Iron through the continuous breeding programme, cultivation of these genotypes after systematic evaluation is expected to benefit in several ways, including the alleviation of malnutrition.

Conclusion
Our study clearly indicated the existence of adequate genetic variability in all the selected genotypes.This variation could be effectively utilized through the development of viable and appropriate breeding strategies for the improvement of rice varieties.The higher estimates of heritability and genetic advance were the significant results of our study, indicating the predominance and frequent occurrence of additive gene action, providing an opportunity for direct selection based on the observed traits.
The phenotypic correlation analysis showed most of the traits evaluated are essential for the selection of high-yielding genotypes as they share a wide range of genetic variability in the selected genotypes of rice.As the selected varieties are found to have high levels of Zinc and Iron, the present study forms the basis for breeding newer genotypes of rice with improved nutritional values.

Fig. 1 :
Fig. 1: Experimental genotype of rice in the field; a. Plants in the vegetative stage; b.Genotypes under aerobic cultivation method during 50% flowering stage; c.Replicated blocks showing grain maturity; d.Field preparation of wetland; e. Plants at the vegetative stage in the wetland; f.Plants at wetland at grain maturity stage

Table 2 . b. Analysis of variance (mean sum of squares) for yield and its attributing traits under wetland conditions during summer Source of
** Significant at 1%, DFF: Days to 50% flowering; DM: Days to Maturity; PH: Plant height; NT: Number of Tillers; NPT: Number of Productive Tillers, PL: Panicle Length, SW: Shoot Weight, GW: Grain Weight, BM: Bio Mass, SL: Length of the seed, SB: Breadth of the seed, LB Ratio: Ratio of Length and Breadth of the Seed, TW: Test weight of 100 seeds

Table 3 . (a): Estimation of genetic parameters for yield and its attributing traits in different genotypes under aerobic & wetland conditions during summer Traits in varied DFF
SE: Standard Error; Min.Range: Minimum Range; Max Range: Maximum Range; GCV %: Genetic Coefficient Variation percent; PCV %: Phenotypic Coefficient Variation percent; h2 Broad sense: Heritability at Broad sense; GAM %: Genetic advance mean percent 1: Aerobic condition; 2: Wetland condition; DFF: Days to 50% flowering; DM: Days to Maturity; PH: Plant height; NT: Number of Tillers; NPT: Number of Productive Tillers, PL: Panicle Length, SW: Shoot Weight, GW: Grain Weight, BM: Bio Mass, SL: Length of the seed, SB: Breadth of the seed, LB Ratio: Ratio of Length and Breadth of the Seed, TW: Test weight of 100 seeds