Estimating the genetic purity in cytoplasmic male sterile (CMS) lines of Egyptian rice.(Report)

Introduction

Rice (Oryza sativa L.) is the well-known holder of two important titles of one of the most important food crop in the world and a model cereal species. At present the most effective and economic way is to develop and extend super rice varieties or hybrids with wide adaptation and super high yielding potential, which is also a fundamental solution to food security problem and an important way to maintain social stability (Liyun et al., 2007). In 1970, Chinese researchers discovered a male sterile rice plant growing naturally within a population of wild rice (Oryza sativa f. spontanea) on Hainan Island. This plant was called wild rice with abortive pollen (WA) and had a particular cytoplasm. The cytoplasm induces the cytoplasmic male sterility (CMS) through interaction with the cell nucleus (Virmani, 1981). Reliable CMS systems can eliminate laborintensive steps of emasculation and hand pollination in F1 seed production and breeding programs (Newton, 1988). Since rice is strictly a self-pollinated crop, hybrid seed production must be based on male sterility systems. Currently, the most popular male-sterility system in rice is the three-line method is based on cytoplasmic genic male sterility and the fertility restoration system. This method involves three lines--the CMS line (A), cognate iso-nuclear maintainer line (B), and restorer line (R)--for the commercial production of rice hybrids. The seed of the male sterile line is multiplied by crossing A and B lines in an isolation plot. Hybrid seed is produced by crossing the A line with an R line in isolation in another plot. The majority of the rice hybrids that are currently under commercial cultivation in the world derive their cytoplasm from the WA source (Yuan, 1995). However, one of big problems is that there might be some parental seeds in commercial hybrids causing low level of genetic purity of hybrids. Any impurities in the hybrids would reduce the expected yield. It has been estimated that every 1% mixture of female line seed in the hybrid seed results in yield reduction of 100 kg per hectare (Mao et al., 1996). The Indian seed act prescribes that, for hybrid rice, the purity should be 98% (Verma, 1996); while in China it is mandated that the purity of hybrid rice should be at least 96% (Yan, 2000). To ensure the required levels of purity in hybrid seed, the parental lines that are utilized in hybrid seed production should have a very high level of purity (ca. 99%). One of the common admixtures that observed during hybrid seed production is that of maintainer lines with those of the CMS lines. Because these are isonuclear, it is not possible to distinguish between them until they flower (Yashitola et al., 2004). The fingerprinting of rice hybrids and identification of their genetic relationships are very important for plant improvement, variety registration system, DUS (distinctness, uniformity and stability) testing, seed purity testing and the protection of plant variety and breeders' rights. Accordingly, clear-cut identification of elite crop varieties and hybrids is essential for protection and prevention of unauthorized commercial use (Nandakumar et al., 2004). On the other hand, purity of hybrid seeds supplied to farmers must surpass 96% (Ichii et al., 2003). Conventional characterization of hybrids based on specific morphological and agronomic data is time-consuming, restricted to a few characteristics, influenced by environmental condition and inefficient. In contrast, DNA-based markers are highly heritable, available in high numbers, and exhibit enough polymerphism, hence they can be used to discriminate closely related genotypes of a plant (Kumar, 1999; Yashitola et al., 2002; Wang et al., 2005). For this reasons, DNA fingerprinting for cultivar or varietals identification has become an important tool for genetic identification in plant breeding and germplasm management (McGregor et al., 2000). Furthermore, Isozymes have been used in genetics for defining systematic phylogentic relationships and to assess the genetic divergence between taxa (Tanksley and Orton, 1983; Bonnin et al., 1996; Yang et al., 1996). The complementary enzyme bands may be used as one of the biochemical indicators for predicting the genetic purity of CMS line. As the esterase isozyme of the progeny has complementary enzyme bands, which differ in its parents, this characteristic has been used in China to do preliminary evaluation of the purity of hybrid seeds. Many scientists studied the correlation of the esterase isozyme with purity in female parent on the bases of the number of complementary enzyme bands with high activity (Devanand et al., 2000). The present study was carried out for estimation the genetic purity of a CMS line (IR 70368A) with its maintainer (IR 70368B) utilizing morphological, biochemical and molecular characterization including isozymes and PCR techniques.

Materials and methods

Plant material and growth conditions

The best time for rice planting is the periods between April 10th and May 10th. The June is the worse cultivation date and reduces all plant properties and consequently grain yield (Abou khalifa, 2009). Egypt has a Mediterranean climate with a typical seasonal rhythm strongly marked with respect to temperature, precipitation and weather in general, hot summers from mid-May to mid-September and rainy, rather changeable, winters from November to mid-March. The plant materials were sown at the experimental farm and Biotechnology Laboratory of the Rice Research and Training Center, Sakha, Kafrelsheikh, Egypt. The cultivation was carried in a clay soil type along summer season (May of 2005, 2006 and 2007) under conditions of no rainfall and humidity 70 - 80%. Three cytoplasmic male sterile lines wild abortive type (WA); IR 58025A, IR 69625A and IR 70368A were tested with their maintainers to determine their genetic purity. These particular lines were chosen based on studies of heterosis and combining ability of 10 CMS lines and 5 Egyptian testers (restorer) to get useful information for hybrid rice program in Egypt. Among the ten CMS lines, IR 58025A, IR 69625A and IR 70368A were the best general combiners for grain yield. During 2005 season, three periodical sowing dates were applied with 15 days intervals to overcome the differences of heading date among the parental lines. Each line was planted in 4 rows, 5 m length and 20 cm apart between plants and rows under isolation plots. A total of 50 single crosses were made and harvested separately for each CMS line. In seasons 2006 and 2007, about 50 populations for each line under isolation plot were sown in the nursery for identification and after 21 days for multiplication. Five replications were grown in randomized complete block design, each replication consisted of one row for the maintainer (1-50) and one row of [F1.sub.(1-50)] crosses (A / B). Each row was 5 m long and contained 25 individual plants. Seedlings were carefully pulled from the nursery after 30 days from seeding and transferred to the permanent field. Seedlings were handling transplanted in hills at the rate of 1-2 Seedlings/hill.

Morphological analysis

The morphological characterizations were conducted by using 5 replicates for all 50 populations of the CMS line. Analysis was reordered for days to heading (measured as days from date of sowing to the date of the first panicle exsertion), plant height (measured as centimeter from soil surface to the tip of the panicle). Yield and yield component characters were measured according to Donald and Humblin (1976); and Yoshida (1981) including, panicle weight (measured as weight (g) of the main panicle after drying), panicle length (measured as the number of centimeters from the panicle neck to the panicle tip-excluding the awn), grain yield (measured as weight (g) of the grain of the each of individual plant), 100-grain weight (recorded as the weight (g) of 100 random filled grains), seed set (%) (seed set = No. of failed grains/No. of total grains x 100), spikelet fertility (measured as number of grains per bagged panicle). The method given by Singh and Haque (1999) was used for determination of pollen viability.

Biochemical and molecular analysis

Isozyme electrophoresis

Fresh leaves of 21 days old seedling (plumules) were used for isozyme analysis (esterase and peroxidase), 200 mg samples were used for the …