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International Journal of Agriculture Sciences ISSN: 0975-3710&E-ISSN: 0975-9107, Volume 8, Issue 47, 2016, pp.-1987-1992. Available online at


Rice Research Institute, Cuttack, Odisha, India University, Cuttack, Odisha, India *Corresponding Author: [email protected] 2Ravenshaw

Received: June 18, 2016; Revised: June 24, 2016; Accepted: July 01, 2016; Published: October 15, 2016 Abstract- The generation of doubled haploids in anther culture aims to accelerate the acquisition of pure lines within a short span of time. Selection of the desired traits can be done directly from anther culture resulted progeny at early generations. However, there is a possibility of segregation in doubled haploids lines in the future for which this experiment was taken up to determine the agronomic traits, uniformity and stability of the DH lines derived from a popular rice hybrid, BS6444G. The first experiment used 200 DH lines of the first generation of anther culture results (A1) following completely randomized design in two years showed each line has uniform agronomic traits; variation was observed in all 200 DHs. However, based on the better performance, 13 DH lines were selected for further evaluation along with the parent hybrid and two better checks in split plot for four seasons. The results showed that the progenies of individuals of 13 DH lines derived from the second to fifth generations showed no difference between generations for each agronomic trait of the same lines which were stable from generation to generation. Simultaneously, SSR markers also confirmed the stability of all the 13 selected DH lines showing homozygosity in all individual DH populations. Therefore, the selected DH lines could be efficiently used for the successful products in future. The 6 selected DH lines could reach the heterotic level over-yielding (5.4%- 41.9%) than the hybrid and better checks. Keywords- Androgenesis, Doubled haploid, Rice hybrid, SSR. Citation: Naik Nupur, et al., (2016) Development of Promising Doubled Haploids from Indica Rice Hybrid: Evaluation of Uniformity and Stability through Morphological and Molecular Markers. International Journal of Agriculture Sciences, ISSN: 0975-3710 & E-ISSN: 0975-9107, Volume 8, Issue 47, pp.-1987-1992. Copyright: Copyright©2016 Naik Nupur, et al., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Academic Editor / Reviewer: Dr Ali El-saied Sharief Introduction Rice (Oryza sativa L.) is the most important food crop of the world utilized exclusively as human staple food for 50% of the world’s population [1]. More than 2 billion people worldwide depend on rice for their survival [2]. Since World population is increasing rapidly, rice production needs to be increased to meet its demands in the coming years in order to keep pace with increasing population [3]. Rice is also the staple food in India providing food to more than half of the Indian population. India is the second largest producer of rice after China and contributes one fifth of the world’s rice production. However, it does not meet the demand of the country’s food security due to its low productivity though a number of varieties are grown in India. In recent years, considerable efforts have been directed towards the improvement of the important agronomic traits of rice from which hybrid rice technology is a feasible and readily adaptable approach showing 1520% yield increase as compared to HYVs. Though utilization of heterosis exhibited by hybrid rice has tremendously enhanced the world productivity [4], however, it is associated by a couple of drawbacks like high seed cost and labour intensive seed production for which intervention of new technology is required to circumvent such type of problems. To overcome these limitations, DH technique is a viable option to generate fertile, stable recombinants from the elite RH as androgenesis has proved its ability to generate homozygous lines in a single generation [5]. Anther culture (AC) technique has proved to be an efficient aid to plant breeding providing a rapid production of doubled haploids (DHs) [6]. Generation of DHs

through AC helps in the rapid fixation of homozygous lines in comparison to conventional breeding which requires 6-7 generations. Doubled haploid techniques not only accelerate the breeding cycle, but also allow better discrimination between genotype within any generation [7]. The populations derived from DHs make genetic studies simple to conduct due to fixation of recessive traits. Androgenic approaches were mostly successful in japonica cultivars [8]. However, the use of AC as a routine technique for breeding of indica rice is extremely limited due to poor induction of androgenic calli and subsequent plant regeneration [9]. The recalcitrance of indica cultivars accounts to low callusing ability, poor plant regeneration potential, and frequent occurrence of albino plant regeneration [10] which limits the practical application for production of homozygous lines. Low androgenic potential in anthers has been fairly overcome with the application of different media combinations [11-13]. In practice Doubled Haploidy technology applied to hybrid rice led to the release of several varieties [14] shows the possibility of fixation of heterosis predominating the partial to complete dominance in DH lines. Though AC is an attractive approach for DHs production, generation of sufficient number of DHs is the prerequisite for using it for practical breeding purposes. Although a number of superior DH rice varieties have been developed from anther culture in China, Japan, Korea and USA [15] with better performance, the reports are meager in terms of the variety release of the developed lines from indica rice hybrids. Thus, the present investigation aimed at studying the association of agronomic characters of DH lines developed from a

International Journal of Agriculture Sciences ISSN: 0975-3710&E-ISSN: 0975-9107, Volume 8, Issue 47, 2016 || Bioinfo Publications ||


Development of Promising Doubled Haploids from Indica Rice Hybrid: Evaluation of Uniformity and Stability through Morphological and Molecular Markers popular rice hybrid, BS6444G for yield improvement along with simultaneous assessment of the genetic stability and homozygosity in individual DH population, which could be utilized as rice varieties.

Hamburg, Germany) programmed for: 4 min at 940C for denaturation followed by 35 cycles of 30 sec at 940C for denaturation, 45 sec at 550C for annealing and 1 min at 720C for polymerization followed by 10 min at 72 0C for final extension. After completion of the PCR, 2.5 µl of 6X loading dye (MBI Ferment Inc., Maryland, USA) was added to the amplified products and were separated in a 2.5% (m/v) agarose (Bangalore Genei, Bangalore, India) gels at 110 V with 1X TBE buffer for 3 hours and stained with EtBr (ethidium bromide). Molecular weight (size) of the amplified products was estimated by using O’Gene Ruler 100 bp DNA ladder (MBI Ferment Inc., Maryland, USA).

Materials and Methods Experimental Design for yield evaluation of selected lines A set of 200 DH lines generated from an F 1 rice hybrid, BS6444G was evaluated under irrigated conditions using an augmented design with 10 blocks each containing 20 test entries and 3 controls (Parent; BS 6444G, Naveen; mid early duration and Tapaswini; medium duration) during the wet season of 2013 and dry season of 2014 at the NRRI experiment field, Cuttack, Odisha, India. Based on these agronomic characters in the field trial, 13 DH lines were selected and evaluated in the field using the randomized complete block experimental design with three replications along with the F1 hybrid and the checks, Naveen and Tapaswini. Thirty days old seedlings raised in wet beds were planted in a well puddled field with a total area of 500 m2 with each DH line planted on a plot size of 10 m2, one-two-plants per hill at spacing of 15 cm x 20 cm. Each plot contained eight rows with 32 plants/row. Recommended doses of N: P: K (90:60:60) were applied in three split doses and need based crop protection measures were undertaken. Weeding was performed by hand and plots were maintained, pest and diseases free until harvest. The observable traits data were collected from initiation of flowering until crop maturity in the replicated individuals. Three successive generations of yield traits were performed from Rabi 2014 to Kharif 2015. The agronomic traits were evaluated based on the following parameters: plant height (cm) measured in from ground level to the top end of the main panicle, days to maturity as the number of days from emergence to yellowish of the panicle at 50% of the plants in a plot, panicle length (cm) from base to tip of the panicle, number of kernels per panicle (fertility %), length and breadth of the flag leaf (cm), the length breadth ratio of the grains. Five panicles selected from five plants chosen randomly were used to determine seed fertility. Seed fertility was computed based on the number of filled spikelets over the total number of spikelets per panicle. Besides, 1000-grain weight (g) and grain yield (t/ ha) were recorded according to the standard evaluation system for rice [16].

Statistical Analysis The data were statistically analyzed using SPAR 2.0 software [18]. The package helped to calculate the means, standard error, standard deviation and coefficient of variations. Frequency distributions were computerized to categorize the DHs into classes. Mean values of 3 replications were calculated for each trait and used in the data analysis. Correlations and path analysis was analysed using Crop Stat 7.2 software (International Rice Research Institute, Philippine). Statistical analyses of the above characters were analyzed [18] for the correlation coefficient. The partitioning of phenotypic correlation coefficient of traits into direct and indirect effects was carried out using the procedure suggested by Dewey and Lu [20] for path analysis. Results Agronomic performance of DHs An efficient androgenic protocol was standardized for the generation of DHs from a popular rice hybrid, BS6444G [Fig-1A–D]. A total of 200 DH lines were assessed in the field for two consecutive seasons based on their agronomic characters [Fig2-A]. Subsequently, 13 DH lines showing the best agronomic performances were selected for further evaluation to check the uniformity and stability. The result showed that 13 DH lines were significantly different from the hybrid along with checks, Naveen and Tapaswini [Fig-2-B] for all the agronomic traits at p

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